Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

ABSTRACT

The present invention relates to a method for identifying a target nucleotide sequence. This method involves forming a ligation product on a target nucleotide sequence in a ligation detection reaction mixture, amplifying the ligation product to form an amplified ligation product in a polymerase chain reaction (PCR) mixture, detecting the amplified ligation product, and identifying the target nucleotide sequence. Such coupling of the ligase detection reaction and the polymerase chain reaction permits multiplex detection of nucleic acid sequence difference.

This application is a continuation of U.S. patent application Ser. No.12/496,447, filed Jul. 1, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/931,403, filed Oct. 31, 2007, now U.S. Pat. No.7,556,924, issued Jul. 7, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/229,366, filed Sep. 16, 2005, now U.S. Pat. No.7,429,453, issued Sep. 30, 2008, which is a continuation of U.S. patentapplication Ser. No. 10/843,720, filed May 12, 2004, now U.S. Pat. No.7,166,434, issued Jan. 23, 2007, which is a continuation of U.S. patentapplication Ser. No. 09/918,156, filed Jul. 30, 2001, now U.S. Pat. No.6,797,470, issued Sep. 28, 2004, which is a continuation of U.S. patentapplication Ser. No. 09/440,523, filed Nov. 15, 1999, now U.S. Pat. No.6,268,148, issued Jul. 31, 2001, which is a divisional of U.S. patentapplication Ser. No. 08/864,473, filed May 28, 1997, now U.S. Pat. No.6,027,889, issued Feb. 22, 2000, and claims the benefit of U.S.Provisional Patent Application Ser. No. 60/018,532, filed May 29, 1996,which are all hereby incorporated by reference in their entirety.

This invention was made with government support under GM41337-06 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to the detection of nucleic acid sequencedifferences using coupled ligase detection reaction (“LDR”) andpolymerase chain reaction (“PCR”). One aspect of the present inventioninvolves use of a ligase detection reaction coupled to a polymerasechain reaction. Another aspect of the present invention relates to theuse of a primary polymerase chain reaction coupled to a secondarypolymerase chain reaction coupled to a ligase detection reaction. Athird aspect of the present invention involves a primary polymerasechain reaction coupled to a secondary polymerase chain reaction.

BACKGROUND OF THE INVENTION Multiplex Detection

Large-scale multiplex analysis of highly polymorphic loci is needed forpractical identification of individuals, e.g., for paternity testing andin forensic science (Reynolds et al., Anal. Chem., 63:2-15 (1991)), fororgan-transplant donor-recipient matching (Buyse et al., TissueAntigens, 41:1-14 (1993) and Gyllensten et al., PCR Meth. Appl, 1:91-98(1991)), for genetic disease diagnosis, prognosis, and pre-natalcounseling (Chamberlain et al., Nucleic Acids Res., 16:11141-11156(1988) and L. C. Tsui, Human Mutat., 1:197-203 (1992)), and the study ofoncogenic mutations (Hollstein et al., Science, 253:49-53 (1991)). Inaddition, the cost-effectiveness of infectious disease diagnosis bynucleic acid analysis varies directly with the multiplex scale in paneltesting. Many of these applications depend on the discrimination ofsingle-base differences at a multiplicity of sometimes closely spacedloci.

A variety of DNA hybridization techniques are available for detectingthe presence of one or more selected polynucleotide sequences in asample containing a large number of sequence regions. In a simplemethod, which relies on fragment capture and labeling, a fragmentcontaining a selected sequence is captured by hybridization to animmobilized probe. The captured fragment can be labeled by hybridizationto a second probe which contains a detectable reporter moiety.

Another widely used method is Southern blotting. In this method, amixture of DNA fragments in a sample is fractionated by gelelectrophoresis, then fixed on a nitrocellulose filter. By reacting thefilter with one or more labeled probes under hybridization conditions,the presence of bands containing the probe sequences can be identified.The method is especially useful for identifying fragments in arestriction-enzyme DNA digest which contains a given probe sequence andfor analyzing restriction-fragment length polymorphisms (“RFLPs”).

Another approach to detecting the presence of a given sequence orsequences in a polynucleotide sample involves selective amplification ofthe sequence(s) by polymerase chain reaction. U.S. Pat. No. 4,683,202 toMullis, et al. and R. K. Saiki, et al., Science 230:1350 (1985). In thismethod, primers complementary to opposite end portions of the selectedsequence(s) are used to promote, in conjunction with thermal cycling,successive rounds of primer-initiated replication. The amplifiedsequence(s) may be readily identified by a variety of techniques. Thisapproach is particularly useful for detecting the presence of low-copysequences in a polynucleotide-containing sample, e.g., for detectingpathogen sequences in a body-fluid sample.

More recently, methods of identifying known target sequences by probeligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M.Whiteley, et al., D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren,et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem.37:1522 (1991). In one approach, known as oligonucleotide ligation assay(“OLA”), two probes or probe elements which span a target region ofinterest are hybridized to the target region. Where the probe elementsbasepair with adjacent target bases, the confronting ends of the probeelements can be joined by ligation, e.g., by treatment with ligase. Theligated probe element is then assayed, evidencing the presence of thetarget sequence.

In a modification of this approach, the ligated probe elements act as atemplate for a pair of complementary probe elements. With continuedcycles of denaturation, hybridization, and ligation in the presence ofpairs of probe elements, the target sequence is amplified linearly,allowing very small amounts of target sequence to be detected and/oramplified. This approach is referred to as ligase detection reaction.When two complementary pairs of probe elements are utilized, the processis referred to as the ligase chain reaction which achieves exponentialamplification of target sequences. F. Barany, “Genetic Disease Detectionand DNA Amplification Using Cloned Thermostable Ligase,” Proc. Nat'lAcad. Sci. USA, 88:189-93 (1991) and F. Barany, “The Ligase ChainReaction (LCR) in a PCR World,” PCR Methods and Applications, 1:5-16(1991).

Another scheme for multiplex detection of nucleic acid sequencedifferences is disclosed in U.S. Pat. No. 5,470,705 to Grossman et. al.where sequence-specific probes, having a detectable label and adistinctive ratio of charge/translational frictional drag, can behybridized to a target and ligated together. This technique was used inGrossman, et. al., “High-density Multiplex Detection of Nucleic AcidSequences: Oligonucleotide Ligation Assay and Sequence-codedSeparation,” Nucl. Acids Res. 22(21):4527-34 (1994) for the large scalemultiplex analysis of the cystic fibrosis transmembrane regulator gene.

Jou, et. al., “Deletion Detection in Dystrophia Gene by Multiplex GapLigase Chain Reaction and Immunochromatographic Strip Technology,” HumanMutation 5:86-93 (1995) relates to the use of a so called “gap ligasechain reaction” process to amplify simultaneously selected regions ofmultiple exons with the amplified products being read on animmunochromatographic strip having antibodies specific to the differenthaptens on the probes for each exon.

There is a growing need (e.g., in the field of genetic screening) formethods useful in detecting the presence or absence of each of a largenumber of sequences in a target polynucleotide. For example, as many as400 different mutations have been associated with cystic fibrosis. Inscreening for genetic predisposition to this disease, it is optimal totest all of the possible different gene sequence mutations in thesubject's genomic DNA, in order to make a positive identification of“cystic fibrosis”. It would be ideal to test for the presence or absenceof all of the possible mutation sites in a single assay. However, theprior-art methods described above are not readily adaptable for use indetecting multiple selected sequences in a convenient, automatedsingle-assay format.

Solid-phase hybridization assays require multiple liquid-handling steps,and some incubation and wash temperatures must be carefully controlledto keep the stringency needed for single-nucleotide mismatchdiscrimination. Multiplexing of this approach has proven difficult asoptimal hybridization conditions vary greatly among probe sequences.

Developing a multiplex PCR process that yields equivalent amounts ofeach PCR product can be difficult and laborious. This is due tovariations in the annealing rates of the primers in the reaction as wellas varying polymerase extension rates for each sequence at a given Mg2+concentration. Typically, primer, Mg2+, and salt concentrations, alongwith annealing temperatures are adjusted in an effort to balance primerannealing rates and polymerase extension rates in the reaction.Unfortunately, as each new primer set is added to the reaction, thenumber of potential amplicons and primer dimers which could formincrease exponentially. Thus, with each added primer set, it becomesincreasingly more difficult and time consuming to work out conditionsthat yield relatively equal amounts of each of the correct products.

Allele-specific PCR products generally have the same size, and an assayresult is scored by the presence or absence of the product band(s) inthe gel lane associated with each reaction tube. Gibbs et al., NucleicAcids Res., 17:2437-2448 (1989). This approach requires splitting thetest sample among multiple reaction tubes with different primercombinations, multiplying assay cost. PCR has also discriminated allelesby attaching different fluorescent dyes to competing allelic primers ina single reaction tube (F. F. Chehab, et al., Proc. Natl. Acad. Sci.USA, 86:9178-9182 (1989)), but this route to multiplex analysis islimited in scale by the relatively few dyes which can be spectrallyresolved in an economical manner with existing instrumentation and dyechemistry. The incorporation of bases modified with bulky side chainscan be used to differentiate allelic PCR products by theirelectrophoretic mobility, but this method is limited by the successfulincorporation of these modified bases by polymerase, and by the abilityof electrophoresis to resolve relatively large PCR products which differin size by only one of these groups. Livak et al., Nucleic Acids Res.,20:4831-4837 (1989). Each PCR product is used to look for only a singlemutation, making multiplexing difficult.

Ligation of allele-specific probes generally has used solid-phasecapture (U. Landegren et al., Science, 241:1077-1080 (1988); Nickersonet al., Proc. Natl. Acad. Sci. USA, 87:8923-8927 (1990)) orsize-dependent separation (D. Y. Wu, et al., Genomics, 4:560-569 (1989)and F. Barany, Proc. Natl. Acad. Sci., 88:189-193 (1991)) to resolve theallelic signals, the latter method being limited in multiplex scale bythe narrow size range of ligation probes. Further, in a multiplexformat, the ligase detection reaction alone cannot make enough productto detect and quantify small amounts of target sequences. The gap ligasechain reaction process requires an additional step—polymerase extension.The use of probes with distinctive ratios of charge/translationalfrictional drag for a more complex multiplex will either require longerelectrophoresis times or the use of an alternate form of detection.

The need thus remains for a rapid single assay format to detect thepresence or absence of multiple selected sequences in a polynucleotidesample.

Microsatellite Analysis

Tandem repeat DNA sequences known as microsatellites represent a verycommon and highly polymorphic class of genetic elements within the humangenome. These microsatellite markers containing small repeat sequenceshave been used for primary gene mapping and linkage analysis. Weber, J.L. et al., Am. J. Hum. Genet. 44: 388-396 (1989); Weissenbach, J. etal., Nature (London) 359: 794-800 (1992). PCR amplification of theserepeats allows rapid assessment for loss of heterozygosity and cangreatly simplify procedures for mapping tumor suppressor genes. Ruppert,J. M., et al., Cancer Res. 53: 5093-94 (1993); van der Riet, et al.,Cancer Res. 54: 1156-58 (1994); Nawroz, H., et al., Cancer Res. 54:1152-55 (1994); Cairns, P., et al., Cancer Res. 54: 1422-24 (1994). Morerecently, they have been used to identify specific mutations in certaininherited disorders including Huntington disease, fragile X syndrome,myotonic dystrophy, spinocerebellar ataxia type I, spinobulbar muscularatrophy, and hereditary dentatorubral-pallidoluysian atrophy. TheHuntington's Disease Collaborative Research Group Cell 72: 971-83(1993); Kremer, E. J., et al., Science 252: 1711-14 (1991); Imbert, G.,et al., Nat. Genet. 4: 72-76 (1993); Orr, H. T., et al., Nat. Genet. 4:221-226 (1993); Biancalana, V., et al., Hum. Mol. Genet. 1: 255-258(1992); Chung, M.-Y., et al., Nat. Genet. 5: 254-258 (1993); Koide, R.,et al., Nat. Genet. 6: 9-13 (1994). These inherited disorders appear toarise from the expansion of trinucleotide repeat units withinsusceptible genes. A more widespread microsatellite instability,demonstrated by expansion or deletion of repeat elements in neoplastictissues, was first reported in colorectal tumors. Peinado, M. A., et al.Proc. Natl. Acad. Sci. USA 89: 10065-69 (1992); Ionov, Y., Nature(London) 363: 558-61 (1993); Thibodeau, S, N., et al., Science 260:816-819 (1993) and later in several other tumor types (Risinger, J. I.,Cancer Res. 53: 5100-03 (1993); Han, H.-J., et al., Cancer Res. 53:5087-89 (1993); Peltomäki, P., Cancer Res. 53: 5853-55 (1993);Gonzalez-Zulueta, M., et al., Cancer Res. 53: 5620-23 (1993); Merlo, A.,et al., Cancer Res. 54: 2098-2101 (1994)). In hereditary nonpolyposiscolorectal carcinoma patients, this genetic instability is apparentlydue to inherited and somatic mutations in mismatch repair genes. Leach,F., et al., Cell 75: 1215-1225 (1993); Fishel, R., et al., Cell 75:1027-38 (1993); Papadopoulos, N., et al., Science 263: 1625-29 (1994);Bronner, C. E., et al., Nature (London) 368: 258-61 (1994).

PCR is commonly used for microsatellite analysis in identifying both theappearance of new polymorphisms and the loss of heterozygosity in cancerdetection. L. Mao, et. al., “Microsatellite Alterations as ClonalMarkers for the Detection of Human Cancer,” Proc. Nat'l Acad. Sci. USA91(21): 9871-75 (1994); L. Mao, et. al., “Molecular Detection of PrimaryBladder Cancer by Microsatellite Analysis,” Science 271:659-62 (1996);D. Radford, et. al., “Allelotyping of Ductal Carcinoma in situ of theBreast: Detection of Loci on 8p, 13q, 161, 17p and 17q,” Cancer Res.55(15): 3399-05 (1995). In using PCR for such purposes, each PCRreaction is run individually and separated on a sequencing gel.

Although these references demonstrate that PCR has application todiagnosis and prognosis of certain cancers, this type of analysis isdeficient, because it does not permit a high throughput and requiressize separation. In addition, there are problems with PCR slippage,causing researchers to shift to tri-, tetra-, and higher nucleotiderepeat units, making cancer detection more difficult.

Microsatellite markers have also been used for colon cancer detection(L. Cawkwell, et. al., “Frequency of Allele Loss of DCC, p53, RB1, WT1,NF1, NM23, and APC/MCC in Colorectal Cancer Assayed by FluorescentMultiplex Polymerase Chain Reaction,” Br. J. Cancer 70(5): 813-18(1994)) and for genome mapping (P. Reed, et. al., “Chromosome-specificMicrosatellite Sets for Fluorescent-Based, Semi-Automated GenomeMapping,” Nat. Genet. 7(3): 390-95 (1994)). However, the key to suchmultiplex processes is the ability to perform them in a single reactiontube. Conventional multiplex microsatellite marker approaches requirecareful attention to primer concentrations and amplification conditions.Although PCR products can be pooled in sets, this requires a prerun onagarose gels to insure that the mixture has about equal amounts of DNAin each band.

Human Identification

PCR has also been used for human identification, such as paternitytesting, criminal investigations, and military personnel identification.A. Syvanen et. al., “Identification of Individuals by Analysis ofBiallelic DNA Markers, Using PCR and Solid-Phase Mini-Sequencing” Am. J.Hum. Genet. 52(1): 46-59 (1993) describes a mini-sequencing approach tohuman identification. The technique requires PCR amplification ofindividual markers with at most 4 PCR reactions being carried out at atime in a single PCR tube. Mini-sequencing is carried out to determineindividual polymorphisms.

Coupled Processes

G. Deng, et. al., “An Improved Method of Competitive PCR forQuantitation of Gene Copy Number,” Nucl. Acids Res. 21: 4848-49 (1993)describes a competitive PCR process. Here, two PCR steps are utilizedwith different sets of primers being used for each gene and itsequivalent standard.

T. Msuih, et. al., “Novel, Ligation-Dependent PCR Assay for Detection ofHepatitis C. Virus in Serum,” J. Clin Microbio. 34: 501-07 (1996) and Y.Park, et. al., “Detection of HCV RNA Using Ligation-Dependent PolymeraseChain Reaction in Formalin-Fixed Paraffin-Embedded Liver Tissue”(submitted) describe the use of a LDR/PCR process in work with RNA.

SUMMARY OF THE INVENTION

The present invention is directed to the detection of nucleic acidsequence differences using coupled LDR and PCR processes. The presentinvention can be carried out in one of the following 3 embodiments: (1)LDR coupled to PCR; (2) primary PCR coupled to secondary PCR coupled toLDR; and (3) primary PCR coupled to secondary PCR. Each of theseembodiments have particular applicability in detecting certaincharacteristics. However, each requires the use of coupled reactions formultiplex detection of nucleic acid sequence differences whereoligonucleotides from an early phase of each process contain sequenceswhich may be used by oligonucleotides from a later phase of the process.

I. Primary PCR/Secondary PCR/LDR Process

One aspect of the present invention relates to a method for identifyingtwo or more of a plurality of sequences differing by one or moresingle-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. This method involves a firstpolymerase chain reaction phase, a second polymerase chain reactionphase, and a ligase detection reaction phase. This process involvesanalyzing a sample potentially containing one or more target nucleotidesequences with a plurality of sequence differences.

In the first polymerase chain reaction phase, one or more primaryoligonucleotide primer groups are provided. Each group comprises one ormore primary oligonucleotide primer sets with each set having a firstnucleotide primer, having a target-specific portion and a 5′ upstreamsecondary primer-specific portion, and a second oligonucleotide primer,having a target-specific portion and a 5′ upstream secondaryprimer-specific portion. The first oligonucleotide primers of each setin the same group contain the same 5′ upstream secondary primer-specificportion and the second oligonucleotide primers of each set in the samegroup contain the same 5′ upstream primer-specific portion. Theoligonucleotide primers in a particular set are suitable forhybridization on complementary strands of a corresponding targetnucleotide sequence to permit formation of a polymerase chain reactionproduct. However, there is a mismatch which interferes with formation ofsuch a polymerase chain reaction product when the primaryoligonucleotide primers hybridize to any other nucleotide sequence inthe sample. The polymerase chain reaction products in a particular setmay be distinguished from other polymerase chain reaction products inthe same group or groups. The primary oligonucleotide primers, thesample, and the polymerase are blended to form a primary polymerasechain reaction mixture.

The primary polymerase chain reaction mixture is subjected to two ormore polymerase chain reaction cycles involving a denaturationtreatment, a hybridization treatment, and an extension treatment, assubstantially described above. During hybridization, target-specificportions of the primary oligonucleotide primers hybridize to the targetnucleotide sequences. The extension treatment causes hybridized primaryoligonucleotide primers to be extended to form primary extensionproducts complementary to the target nucleotide sequence to which theprimary oligonucleotide primers are hybridized.

Although the upstream secondary primer-specific portions of a primaryoligonucleotide primer set are not present on the target DNA, theirsequences are copied by the second and subsequent cycles of the primarypolymerase chain reaction phase. As a result, the primary extensionproducts produced after the second cycle have the secondaryprimer-specific portions on their 5′ ends and the complement ofprimer-specific portion on their 3′ ends.

Next, there is a second polymerase chain reaction phase. This phaseinvolves providing one or a plurality of secondary oligonucleotideprimer sets. Each set has a first secondary oligonucleotide primercontaining the same sequence as the 5′ upstream portion of the firstprimary oligonucleotide primer, and a second secondary oligonucleotideprimer containing the same sequence as the 5′ upstream portion of thesecond primary oligonucleotide primer from the same primaryoligonucleotide primer set as the first primary oligonucleotidecomplementary to the first secondary primer. A set of secondaryoligonucleotide primers may be used to amplify all of the primaryextension products in a given group. The secondary oligonucleotideprimers are blended with the primary extension products and thepolymerase to form a secondary polymerase chain reaction mixture.

The secondary polymerase chain reaction mixture is subjected to one ormore polymerase chain reaction cycles having a denaturation treatment, ahybridization treatment, and an extension treatment, as substantiallyset forth above. During the hybridization treatment, the secondaryoligonucleotide primers hybridize to the complementary sequences presenton the primary extension products but not to the original targetsequence. The extension treatment causes the hybridized secondaryoligonucleotide primers to be extended to form secondary extensionproducts complementary to the primary extension products.

The last phase of this aspect of the present invention involves a ligasedetection reaction process. Here, a plurality of oligonucleotide probesets are provided where each set has a first oligonucleotide probe,having a secondary extension product-specific portion and a detectablereporter label, and a second oligonucleotide probe, having a secondaryextension product-specific portion. The oligonucleotide probes in aparticular set are suitable for ligation together when hybridizedadjacent to one another on a complementary secondary extensionproduct-specific portion. However, there is a mismatch which interfereswith such ligation when the oligonucleotide probes are hybridized to anyother nucleotide sequence present in the sample. The ligation product ofoligonucleotide probes in a particular set may be distinguished fromeither probe or other ligation products. The plurality ofoligonucleotide probe sets, the secondary extension products, and aligase are blended to form a ligase detection reaction mixture.

The ligase detection reaction mixture is subjected to one or more ligasedetection reaction cycles having a denaturation treatment andhybridization treatment substantially as described above. In thehybridization treatment, the oligonucleotide probe sets hybridize atadjacent positions in a base-specific manner to the respective secondaryextension products if present. As a result, adjacent probes ligate toone another to form a ligation product sequence containing thedetectable reporter label and the secondary extension product-specificportions connected together. The oligonucleotide probe sets mayhybridize to nucleotide sequences other than their respectivecomplementary secondary extension products but do not ligate togetherdue to the presence of one or more mismatches and individually separateduring the denaturation treatment. Following the ligase detectionreaction cycles, the reporter labels of the ligation product sequencesare detected which indicates the presence of one or more targetnucleotide sequences in the sample.

The primary PCR/secondary PCR/LDR process of the present inventionprovides significant advantages over the use of PCR alone in themultiplex detection of single nucleotide and tandem repeatpolymorphisms.

As noted above, the use of PCR alone requires heavy optimization ofoperating conditions in order to conduct multiplex detection procedures.Moreover, the quantity of oligonucleotide primers must be increased todetect greater numbers of target nucleotide sequences. However, as thisoccurs, the probability of target independent reactions (e.g., theprimer-dimer effect) increases. In addition, the mutations must beknown, false positives may be generated by polymerase extension off ofnormal template, closely-clustered sites due to interference ofoverlapping primers cannot undergo multiplex detection, single base orsmall insertions and deletions in small repeat sequences cannot bedetected, and quantification of mutant DNA in high background of normalDNA is difficult. As a result, the number of target nucleotide sequencesdetected in a single multiplex PCR process is limited.

Direct sequencing requires enrichment of mutant samples in order tocorrect sequences, requires multiple reactions for large genescontaining many exons, requires electrophoretic separation of products,is time consuming, and cannot be used to detect mutant DNA in less than5% of background of normal DNA. When mini-sequencing, the mutation mustbe known, closely-clustered sites due to interference of overlappingprimers cannot undergo multiplex detection, single base or smallinsertions and deletions in small repeat sequences cannot be detected,and four separate reactions are required. For allele-specificoligonucleotide hybridization (“ASO”), the mutation must be known,hybridization and washing conditions must be known, cross-reactivity isdifficult to prevent, closely-clustered sites due to interference ofoverlapping primers cannot undergo multiplex detection, and mutant DNAcannot be detected in less than 5% of background of normal DNA.Primer-mediated RFLP requires electrophoretic separation to distinguishmutant from normal DNA, is of limited applicability to sites that may beconverted into a restriction site, requires additional analysis todetermine the nature of the mutation, and is difficult to use where themutant DNA is in a high background of normal DNA. Single strandconformational polymorphism analysis (“SSCP”) requires electrophoreticseparation to distinguish mutant conformer from normal conformer, misses30% of possible mutations, requires additional analysis to determine thenature of the mutation, and cannot distinguish mutations from silentpolymorphisms. With dideoxynucleotide finger printing (“ddF”), it isdifficult to detect mutations in a high background of normal DNA,electrophoretic separation is required to distinguish mutant conformerfrom normal conformer, additional analysis must be used to determine thenature of the mutation, and mutations cannot be distinguished fromsilent polymorphisms. Denaturing gradient gel electrophoresis (“DGGE”)must electrophoretically separate mutant conformer from normalconformer, misses 30% of possible mutations, requires additionalanalysis to determine the nature of the mutation, cannot distinguishmutations from silent polymorphisms, and imposes technical challenges toreproducing previously achieved results. RNase mismatch cleavagerequires additional analysis to determine the nature of the mutation,requires analysis of both strands to exclude RNase-resistant mismatches,and imposes difficulty in detecting mutations in a high background ofnormal DNA. Chemical mismatch cleavage cannot detect mutant DNA in lessthan 5% of background of normal DNA, and requires an analysis of bothstrands to detect all mutations. For T4 Endo VII mismatch cleavage,additional analysis is needed to determine the nature of the mutation,mutations cannot be distinguished from silent polymorphisms,endonuclease cleaves control DNA which necessitates carefulinterpretation of results, and it is difficult to detect mutations in ahigh background of normal DNA.

These problems are avoided in the primary PCR/secondary PCR/LDR processof the present invention which combines the sensitivity of PCR with thespecificity of LDR. The primary PCR phase produces primary extensionproducts with a secondary primer-specific portion. This initial phase iscarried out under conditions effective to maximize production of primaryextension products without obtaining the adverse effects sometimesachieved in a PCR-only process. In particular, the primary PCR phase ofthe present invention is carried out with 15 to 20 PCR cycles andutilizes less primer than would be utilized in a PCR-only process. Theprimary PCR phase of the present invention produces extension productsin a varied and unpredictable way, because some target nucleotidesequences will be amplified well, while others will not. However, in thesecondary PCR phase, all of the primary extension products are amplifiedapproximately equally, because they all have the same secondaryprimer-specific portions. Target nucleotide sequences originally presentin the sample will not be amplified by the secondary PCR phase, becausesuch sequences do not contain a secondary primer-specific portion. As aresult, the primary PCR/secondary PCR/LDR process of the presentinvention is able to achieve multiplex detection of hundreds ofnucleotide sequence differences in a single tube without unduecustomization of operating conditions for each particular sample beinganalyzed. Since the selection of mutant sequences is mediated by LDRrather than PCR, the primary PCR/secondary PCR/LDR process is lesssusceptible to false-positive signal generation. In addition, theprimary PCR/secondary PCR/LDR process allows detection ofclosely-clustered mutations, detection of single base or smallinsertions and deletions in small repeat sequences, quantitativedetection of less than 1% mutations in high background of normal DNA,and detection of ligation product sequences using addressable arrays.The only significant requirements are that the mutations be known andthat a multitude of oligonucleotides be synthesized.

The ability to detect single nucleotide and tandem repeat polymorphismsis particularly important for forensic DNA identification and diagnosisof genetic diseases.

II. LDR/PCR Process

A second aspect of the present invention relates to a method foridentifying one or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. This method has a ligasedetection reaction phase followed by a polymerase chain reaction phase.This method involves providing a sample potentially containing one ormore target nucleotide sequences with a plurality of sequencedifferences.

In the ligase detection reaction phase, one or more oligonucleotideprobe sets are provided. Each set has a first oligonucleotide probe,having a target-specific portion and a 5′ upstream primer-specificportion, and a second oligonucleotide probe, having a target-specificportion and a 3′ downstream primer-specific portion. The oligonucleotideprobes in a particular set are suitable for ligation together whenhybridized adjacent to one another on a corresponding target nucleotidesequence. However, there is a mismatch which interferes with suchligation when they are hybridized to any other nucleotide sequencepresent in the sample. The sample, the plurality of oligonucleotideprobe sets, and a ligase are blended together to form a ligase detectionreaction mixture.

The ligase detection reaction mixture is subjected to one or more ligasedetection reaction cycles. These cycles include a denaturation treatmentand a hybridization treatment. In the denaturation treatment, anyhybridized oligonucleotides are separated from the target nucleotidesequences. The hybridization treatment causes the oligonucleotide probesets to hybridize at adjacent positions in a base-specific manner totheir respective target nucleotide sequences if present in the sample.Once hybridized, the oligonucleotide probe sets ligate to one another toform a ligation product sequence. This product contains the 5′ upstreamprimer-specific portion, the target-specific portions connectedtogether, and the 3′ downstream primer-specific portion. The ligationproduct sequence for each set is distinguishable from other nucleicacids in the ligase detection reaction mixture. The oligonucleotideprobe sets hybridized to nucleotide sequences in the sample other thantheir respective target nucleotide sequences but do not ligate togetherdue to a presence of one or more mismatches and individually separateduring the subsequent denaturation treatment.

In the polymerase chain reaction, one or a plurality of oligonucleotideprimer sets are provided. Each set has an upstream primer containing thesame sequence as the 5′ upstream primer-specific portion of the ligationproduct sequence and a downstream primer complementary to the 3′downstream primer-specific portion of the ligation product sequence,where one primer has a detectable reporter label. The ligase detectionreaction mixture is blended with the one or a plurality ofoligonucleotide primer sets and the polymerase to form a polymerasechain reaction mixture.

The polymerase chain reaction mixture is subjected to one or morepolymerase chain reaction cycles which include a denaturation treatment,a hybridization treatment, and an extension treatment. During thedenaturation treatment, hybridized nucleic acid sequences are separated.The hybridization treatment causes primers to hybridize to theircomplementary primer-specific portions of the ligation product sequence.During the extension treatment, hybridized primers are extended to formextension products complementary to the sequences to which the primersare hybridized. In a first cycle of the polymerase chain reaction phase,the downstream primer hybridizes to the 3′ downstream primer-specificportion of the ligation product sequence and is extended to form anextension product complementary to the ligation product sequence. Insubsequent cycles the upstream primer hybridizes to the 5′ upstreamprimer-specific portion of the extension product complementary to theligation product sequence and the downstream primer hybridizes to the 3′downstream portion of the ligation product sequence.

Following the polymerase chain reaction phase of this process, thereporter labels are detected and the extension products aredistinguished to indicate the presence of one or more target nucleotidesequences in the sample.

One embodiment of the LDR/PCR process of the present invention achievesimproved results over the use of LDR alone in measuring the number ofgene copies in a cell (i.e. gene dosage). When LDR alone is utilized, itis difficult to produce sufficient target copies which are neededultimately to quantify a plurality of genes.

In another embodiment of the LDR/PCR process of the present invention,the LDR phase ligation product sequences are produced in a ratioproportional to the ratio of the genes from which they were derivedwithin the sample. By incorporation of the same primer-specific portionsin the oligonucleotide probes for the LDR phase, the PCR phase amplifiesligation product sequences to the same degree so that theirproportionality is maintained. Target sequences originally found in thesample being analyzed are not amplified by the PCR phase, because suchtarget sequences do not contain PCR primer-specific portions. Inaddition, since only the oligonucleotide primers for the PCR phase havereporter labels, only extension products with those labels will bedetected.

Determination of variation in gene dosage is important in a number ofbiomedical applications.

Males differ in gene dosage from females for those genes located on theX chromosome, women having two copies while men have one copy. Women maybe carriers of deletions along the X chromosome. If the deleted regionof the X chromosome included one or more genes, then the woman has onlyone copy of these genes in the corresponding, non-deleted area of theother X chromosome. The result (having one copy of an X-linked gene) issimilar to the situation in male cells and is usually tolerated withoutmanifestations of an inherited disorder. However, if the woman's soninherits her deleted X chromosome, he will have no copies of the genesin the deletion region and suffer from the X-linked disorder related tothe absence of the gene. The detection of chromosomal deletions,therefore, is one application of the LDR/PCR process of the presentinvention.

Congenital chromosomal disorders occur when a fertilized egg has anabnormal compliment of chromosomes. The most common congenitalchromosomal disorder is Down Syndrome, which occurs when there is anadditional chromosome 21 in each cell, designated 47,XX+21 or 47,XY+21.The LDR/PCR process of the present invention can be designed to identifycongenital chromosomal disorders.

The LDR/PCR process of the present invention is also useful fordistinguishing polymorphisms in mono-nucleotide and di-nucleotide repeatsequences. It will also be useful in distinguishing minor populations ofcells containing unique polymorphisms for detection of clonality.

The LDR/PCR process can be used with both gel and non-gel (i.e. DNAarray) technologies. It allows multiplex analysis of many geneamplifications and deletions simultaneously, allows quantitativeanalysis, and does not require an external standard. The only relativelyminor challenge presented by the LDR/PCR process is that it is difficultto use in determining the boundaries of large deletions in chromosomes.

By contrast, microsatellite marker analysis cannot be used to detectsmall regions that are deleted or amplified, is not compatible withsimultaneous detection of amplified regions, and depends on theavailability of informative markers. Competitive PCR (i.e. differentialPCR) cannot be used in a multiplex format to detect several deletionsand amplifications simultaneously, and is not particularly accurate.Southern hybridization is time consuming, labor intensive, is notamenable to multiplexing due to the need for multiple steps for eachprobe tested, and requires large quantities of DNA. Fluorescent in situhybridization (“FISH”) requires specialized expertise, is timeconsuming, and requires large probes to analyze for suspected deleted oramplified regions. Thus, the LDR/PCR process of the present inventionconstitutes a significant advance over prior processes.

III. Primary PCR/Secondary PCR Process

A third aspect of the present invention also involves a method foridentifying two or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations inone or more target nucleotide sequences. This method involves subjectinga sample potentially containing one or more target nucleotide sequenceswith a plurality of sequence differences to two successive polymerasechain reaction phases.

For the first polymerase chain reaction phase, one or more primaryoligonucleotide primer groups are provided where each group comprisestwo or more primary oligonucleotide primer sets. Each set has a firstoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primary-specific portion, and a secondoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primer-specific portion. The first oligonucleotideprimers of each set in the same group contain the same 5′ upstreamsecondary primer-specific portion and the second oligonucleotide primersof each set in the same group contain the same 5′ upstream secondaryprimer-specific portion. The oligonucleotide primers in a particular setare suitable for hybridization on complementary strands of acorresponding target nucleotide sequence to permit formation of apolymerase chain reaction product. However, there is a mismatch whichinterferes with formation of such a polymerase chain reaction productwhen the primary oligonucleotide primers hybridize to any othernucleotide sequence present in the sample. The polymerase chain reactionproducts in a particular set may be distinguished from other polymerasechain reaction products with the same group or other groups. The primaryoligonucleotide primers are blended with the sample and the polymeraseto form a primary polymerase chain reaction mixture.

The primary polymerase chain reaction mixture is subjected to two ormore polymerase chain reaction cycles involving a denaturationtreatment, a hybridization treatment, and an extension treatment, asdescribed above. During the hybridization treatment, the target-specificportion of a primary oligonucleotide primer is hybridized to the targetnucleotide sequences. In the extension treatment, the hybridized primaryoligonucleotide primers are extended to form primary extension productscomplementary to the target nucleotide sequence to which the primaryoligonucleotide primer is hybridized.

Although the upstream secondary primer-specific portions of a primaryoligonucleotide primer set are not present on the target DNA, theirsequences are copied by the second and subsequent cycles of the primarypolymerase chain reaction phase. As a result, the primary extensionproducts produced after the second and subsequent cycles have thesecondary primer-specific portions on their 5′ ends and the complementof primer-specific portion on their 3′ ends.

In the second polymerase chain reaction phase of this aspect of thepresent invention, one or a plurality of secondary oligonucleotideprimer sets are provided. Each set has a first secondary primer having adetectable reporter label and containing the same sequence as the 5′upstream portion of a first primary oligonucleotide primer, and a secondsecondary primer containing the same sequence as the 5′ upstream primerof the second primary oligonucleotide primer from the same primaryoligonucleotide primer set as the first primary oligonucleotidecomplementary to the first secondary primer. A set of secondaryoligonucleotide primers amplify the primary extension products in agiven group. The secondary oligonucleotide primers are blended with theprimary extension products and the polymerase to form a secondarypolymerase chain reaction mixture.

The secondary polymerase chain reaction mixture is subjected to one ormore polymerase chain reaction cycles involving a denaturationtreatment, a hybridization treatment, and an extension treatment, asdescribed above. In the hybridization treatment, the secondaryoligonucleotide primers are hybridized to the primary extensionproducts, while the extension treatment causes the hybridized secondaryoligonucleotide primers to be extended to form secondary extensionproducts complementary to the primary extension products. Aftersubjecting the secondary polymerase chain reaction mixture to the two ormore polymerase chain reaction cycles, the labelled secondary extensionproducts are detected. This indicates the presence of one or more targetnucleotide sequences in the sample.

The primary PCR/secondary PCR process of the present invention providessignificant advantages over Southern hybridization, competitive PCR, andmicrosatellite marker analysis in detecting nucleotide deletions whichcause a loss of heterozygosity. Although Southern hybridization is moreaccurate than competitive PCR, it is quite labor intensive, requireslarge amounts of DNA, and neither technique can be multiplexed. Currentmultiplex microsatellite marker approaches require careful attention toprimer concentrations and amplification conditions.

The primary PCR/secondary PCR process of the present invention overcomesthese difficulties encountered in the prior art. In the primary PCRphase, the primary oligonucleotide primers flank dinucleotide or otherrepeat sequences and include a secondary primer-specific portion. Theprimary PCR phase is carried out at low concentrations of these primersto allow several loci to undergo amplification at the same time. Thesecondary PCR phase causes amplification to continue at the same ratewith target-specific secondary primers being selected to space one setof microsatellite markers from the adjacent set. The primaryPCR/secondary PCR process can be used to carry out multiplex detectionin a single PCR tube and with single gel lane analysis.

This aspect of the present invention is useful in carrying out amicrosatellite marker analysis to identify nucleotide deletions in agene. Such multiplex detection can be carried out in a single reactiontube. However, the primary PCR/secondary PCR process cannot distinguishamplifications from deletions, so, when making such distinctions, thisprocess must be used in conjunction with the above-described LDR/PCRprocess or a differential PCR process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of germline mutations, such as point mutations, byelectrophoresis or capture on an addressable array. Note that the term“zip-code” which appears in FIG. 1 and other drawings refers to asequence specific to a subsequently used primer or probe but not toeither the target sequence or other genome sequences.

FIG. 2 is a flow diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of biallelic polymorphisms by electrophoresis orcapture on an addressable array.

FIG. 3 is a flow diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of cancer-associated mutations by electrophoresisor capture on an addressable array.

FIG. 4 is a schematic diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of biallelic polymorphisms.

FIG. 5 is a schematic diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of allelic differences using LDR oligonucleotideprobes which distinguish all possible bases at a given site.

FIG. 6 is a schematic diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of the presence of any possible base at two nearbysites using LDR oligonucleotide probes which distinguish all possiblebases at a given site.

FIG. 7 is a schematic diagram depicting a primary PCR/secondary PCR/LDRprocess for detection of cancer-associated mutations at adjacentalleles.

FIG. 8 is a flow diagram depicting an LDR/PCR process with and withoutrestriction endonuclease digestion using electrophoresis detection.

FIG. 9 is a flow diagram depicting an LDR/PCR process using detection onan addressable array using gene-specific addresses.

FIG. 10 is a schematic diagram depicting an LDR/PCR process formultiplex detection of gene amplifications and deletions.

FIG. 11 is a schematic diagram depicting an allele specific problem foran LDR/PCR process.

FIG. 12 is a schematic diagram depicting a solution for the allelespecific problem for an LDR/PCR process which is shown in FIG. 11.

FIG. 13 is a flow diagram depicting an LDR/PCR process with anintermediate exonuclease digestion phase for detection of biallelicpolymorphisms by electrophoresis or capture on an addressable array.

FIG. 14 is a flow diagram depicting an LDR/PCR process with anintermediate exonuclease digestion phase for detection ofcancer-associated mutations by electrophoresis or capture on anaddressable array.

FIG. 15 is a schematic diagram depicting an LDR/PCR process with anintermediate exonuclease digestion phase for detection of allelespecific mutations and polymorphisms.

FIG. 16 is a schematic diagram depicting an LDR/PCR process with anintermediate exonuclease digestion phase for detection of mononucleotiderepeat polymorphisms.

FIG. 17 is a schematic diagram depicting an LDR/PCR process with anintermediate exonuclease digestion phase for detection of mononucleotiderepeat polymorphisms which are in low abundance.

FIG. 18 is a flow diagram depicting the detection of polymorphisms usingan LDR/PCR process with an intermediate sequenase extension phase and auracil N-glycosylase digestion phase after the LDR phase and before thePCR phase and with detection by electrophoresis or an addressable array.

FIG. 19 is a flow diagram depicting the detection of cancer using anLDR/PCR process with an intermediate sequenase extension phase and auracil N-glycosylase digestion phase after the LDR phase and before thePCR phase and with detection by electrophoresis or an addressable array.

FIG. 20 is a schematic diagram depicting detection of mononucleotiderepeats using an LDR/PCR process with an intermediate sequenaseamplification phase and a uracil N-glycosylase digestion phase after theLDR phase and before the PCR phase.

FIG. 21 is a schematic diagram depicting detection of mononucleotiderepeat polymorphisms which are in low abundance using an LDR/PCR processwith an intermediate sequenase amplification phase and a uracilN-glycosylase digestion phase after the LDR phase and before the PCRphase.

FIG. 22 is a flow diagram depicting a primary PCR/secondary PCR processfor detection of microsatellite repeats.

FIG. 23 is a schematic diagram depicting a primary PCR/secondary PCRprocess for multiplex detection of insertions and deletions inmicrosatellite repeats.

FIG. 24 shows the design of LDR oligonucleotide probes forquantification of gene amplifications and deletions in an LDR/PCRprocess.

FIGS. 25A-D show electropherogram results for an LDR/PCR process.

FIGS. 26A-C show electropherogram results for an LDR/PCR process ofErbB, G6PD, Int2, p53, and SOD gene segments from normal human femaleDNA and from DNA of the breast cancer cell line ZR-75-30 and the gastriccarcinoma cell line SKGT-2. The ErbB gene is known to be amplified inthe cancer cell lines. Target-specific ligation product sequences of 104bp are generated in 10 cycles (94° C. for 30 sec, 65° C. for 4 min) ofLDR using 500 femtomoles of each ligation primer, 50 ng of genomic DNA,124 units of Thermus thermophillus (“Tth”) ligase, 2 μl of 10× buffer(0.2 M Tris, pH 8.5 and 0.1 M MgCl₂), 2 μl of 10 mM NAD, and 1 μl of 200mM DTT in a volume of 20 μl. The ligation products are proportionallyamplified in 26 cycles (94° C. for 15 sec, 60° C. for 50 sec) of PCR bythe addition of 30 μl of a solution containing 5 μl 10× Stoffel buffer(Perkin Elmer), 25 picomoles of each oligonucleotide primer, 2.5 unitsof Tag polymerase Stoffel fragment, and 8 μl of a solution 5 mM in eachdNTP. After amplification, the products are digested with HaeIII andHinP1I to generate FAM-labeled products of 58 bp (ErbB (i.e.HER-2/neu/erbB oncogene)), 61 bp (G6PD), 67 bp (Int2 (i.e. int-2oncogene)), 70 bp (p53) and 76 bp (SOD). These products are separatedand analyzed on a 373A DNA sequencer using the Genescan 672 softwarepackage (Applied Biosystems, Inc., Foster City, Calif.). Results aredisplayed as electropherograms such that peak heights and areas reflectthe amounts of the PCR products. In FIG. 26A, the gene dosagedetermination for the five loci in normal human female DNA is shown. Thepeak heights and areas for G6PD, Int2, p53, and SOD are very similar.The peak height and area for ErbB is consistently small in normalgenomic DNA. In FIG. 26B, the peak height and area for ErbB are elevatedwhen gene dosage is investigated in ZR-75-30, a cell line with knownErbB amplification. In FIG. 26C, the gastric cell line, SKGT-2 showsdramatic amplification of the ErbB gene and a modest amplification ofInt2. The G6PD gene peak may be embedded in the large ErbB peak.

FIGS. 27A-C show electropherogram results for an LDR/PCR process todetermine whether amplification of ErbB affected the relative peakheights of the other LDR oligonucleotide probes and PCR oligonucleotideprimers for G6PD, Int2, p53, and SOD. In FIG. 27A, the gene dosagedetermination for the four loci in normal human female DNA is shown.Peak heights and areas for G6PD, Int2, p53, and SOD are similar, as theywere in the experiment using all five LDR primers. In FIG. 27B, G6PD,Int2, and SOD analyzed in the ZR-75-30 breast cancer cell line showsimilar relative peak heights, comparable to their appearance in normalfemale DNA. The peak height for p53 is reduced, suggesting the deletionof this gene in a portion of the cells in this cell line. In FIG. 27C,in the gastric carcinoma cell line, SKGT-2, G6PD, and p53 showcomparable peak heights. The Int2 peak height remains relatively high,as it was in the experiment using all five LDR oligonucleotide probes.Thus, the LDR and PCR amplification of each product appears to beindependent of the other products during the course of the experiment.

FIGS. 28A-C show electropherogram results for the PCR phase of a primaryPCR/secondary PCR/LDR process. Multiplex PCR amplification of 12 lociusing primary PCR oligonucleotide primers produces approximately equalamounts of product. Over 80 gene regions with single-base polymorphismswere identified from the Human Genome Database. Twelve of these (seeTable 10 and FIGS. 29A-H) were amplified in a primary PCR phase asfollows: Long primary PCR oligonucleotide primers were designed to havegene-specific 3′ ends and 5′ ends complementary to one of two sets ofsecondary PCR oligonucleotide primers. The upstream primary PCRoligonucleotide primers were synthesized with either FAM (i.e.6-carboxyfluorescein; fluorescent dye used in sequencing and mutationdetection) or TET (i.e. tetrachlorinated-6-carboxyfluorescein;fluorescent dye used in sequencing/mutation detection) fluorescentlabels. All 24 base long primary PCR oligonucleotide primers were usedat low concentration (2 picomole of each primer in 20 μl) in a 15 cycleprimary PCR phase. After this, the two sets of secondary PCRoligonucleotide primers were added at higher concentrations (25picomoles of each) and the secondary PCR phase was conducted for anadditional 25 cycles. The products were separated on a 373 DNA Sequencer(Applied Biosystems). Panel A shows the electropherogram results for theFAM- and TET-labelled products combined. Panel B shows the FAM-labelledproducts alone. Panel C shows the TET-labelled products alone. Theprocess produces similar amounts of multiplexed products without theneed to adjust carefully primer concentrations or PCR conditions.

FIGS. 29A-H show electropherogram results for the LDR phase of a primaryPCR/secondary PCR/LDR process in detecting 12 biallelic genes forforensic identification. The primary and secondary PCR phases for the 12polymorphic genes were performed as described in FIG. 28A-C. However,the secondary PCR oligonucleotide primers were not fluorescentlylabelled. The secondary PCR process extension products were diluted intoa ligase buffer containing 36 LDR oligonucleotide probes (one common andtwo discriminating primers for each locus). LDR oligonucleotide probesets were designed in two ways: (i) allele-specific oligonucleotideprobes were of the same length but contained either the FAM or TETlabel; or (ii) the allele-specific oligonucleotide probes were bothlabelled with HEX (i.e. hexachlorinated-6-carboxyfluorescein;fluorescent dye used in sequencing and mutation detection) but differedin length by two basepairs. After 20 cycles of the LDR phase, theligation product sequences were resolved using a 10% polyacrylamidesequencing gel on a 373 DNA Sequencer (Applied Biosystems). Panel A andE show the 12 loci PCR/LDR profiles of two individuals. Panels B, C, andD show, respectively, the FAM, TET, and HEX data for the individual inpanel A. Panels F, G, and H show, respectively, the FAM, TET, and HEXdata for the individual in panel E. The individual in panel A ishomozygous only at locus 6 (ALDOB (i.e. aldolase B)) and locus 8 (IGF(i.e. insulin growth factor)). The individual in panel E is heterozygousonly at loci 3 (C6 (i.e. complement component C6)), 5 (NF1 (i.e.neurofibromatosis)), 6 (ALDOB), and 8 (IGF). This demonstrates that theprimary PCR/primary PCR/LDR process can simultaneously distinguish bothhomozygous and heterozygous genotypes at multiple positions.

DETAILED DESCRIPTION OF THE INVENTION I. Primary PCR/Secondary PCR/LDRProcess

One aspect of the present invention relates to a method for identifyingtwo or more of a plurality of sequences differing by one or moresingle-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. This method involves a firstpolymerase chain reaction phase, a second polymerase chain reactionphase, and a ligase detection reaction phase. This process involvesanalyzing a sample potentially containing one or more target nucleotidesequences with a plurality of sequence differences.

In the first polymerase chain reaction phase, one or more primaryoligonucleotide primer groups are provided. Each group comprises one ormore primary oligonucleotide primer sets with each set having a firstoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primer-specific portion, and a second oligonucleotideprimer, having a target-specific portion and a 5′ upstream secondaryprimer-specific portion. The first oligonucleotide primers of each setin the same group contain the same 5′ upstream secondary primer-specificportion and the second oligonucleotide primers of each set in the samegroup contain the same 5′ upstream primer-specific portion. Theoligonucleotide primers in a particular set are suitable forhybridization on complementary strands of a corresponding targetnucleotide sequence to permit formation of a polymerase chain reactionproduct. However, there is a mismatch which interferes with formation ofsuch a polymerase chain reaction product when the primaryoligonucleotide primers hybridize to any other nucleotide sequencepresent in the sample. The polymerase chain reaction products in aparticular set may be distinguished from other polymerase chain reactionproducts in the same group or groups. The primary oligonucleotideprimers, the sample, and the polymerase are blended to form a primarypolymerase chain reaction mixture.

The primary polymerase chain reaction mixture is subjected to two ormore polymerase chain reaction cycles involving a denaturationtreatment, a hybridization treatment, and an extension treatment, assubstantially described above. During hybridization, target-specificportions of the primary oligonucleotide primers hybridize to the targetnucleotide sequences. The extension treatment causes hybridized primaryoligonucleotide primers to be extended to form primary extensionproducts complementary to the target nucleotide sequence to which theprimary oligonucleotide primers are hybridized.

Although the upstream secondary primer-specific portions of a primaryoligonucleotide primer set are not present on the target DNA, theirsequences are copied by the second and subsequent cycles of the primarypolymerase chain reaction phase. As a result, the primary extensionproducts produced after the second cycle have the secondaryprimer-specific portions on their 5′ ends and the complement of theprimer-specific portion on their 3′ ends.

Next, there is a second polymerase chain reaction phase. This phaseinvolves providing one or a plurality of secondary oligonucleotideprimer sets. Each set has a first secondary oligonucleotide primercontaining the same sequence as the 5′ upstream portion of the firstprimary oligonucleotide primer, and a second secondary oligonucleotideprimer containing the same sequence as the 5′ upstream portion of thesecond primary oligonucleotide primer from the same primaryoligonucleotide primer set as the first primary oligonucleotidecomplementary to the first secondary primer. A set of secondaryoligonucleotide primers may be used to amplify all of the primaryextension products in a given group. The secondary oligonucleotideprimers are blended with the primary extension products and thepolymerase to form a secondary polymerase chain reaction mixture.

The secondary polymerase chain reaction mixture is subjected to one ormore polymerase chain reaction cycles having a denaturation treatment, ahybridization treatment, and an extension treatment, as substantiallyset forth above. During the hybridization treatment, the secondaryoligonucleotide primers hybridize to complementary sequences present onthe primary extension products but not to the original target sequence.The extension treatment causes the hybridized secondary oligonucleotideprimers to be extended to form secondary extension productscomplementary to the primary extension products.

The last phase of this aspect of the present invention involves a ligasedetection reaction process. Here, a plurality of oligonucleotide probesets are provided where each set has a first oligonucleotide probe,having a secondary extension product-specific portion and a detectablereporter label, and a second oligonucleotide probe, having a secondaryextension product-specific portion. The oligonucleotide probes in aparticular set are suitable for ligation together when hybridizedadjacent to one another on a complementary secondary extensionproduct-specific portion. However, there is a mismatch which interfereswith such ligation when the oligonucleotide probes are hybridized to anyother nucleotide sequence present in the sample. The ligation product ofoligonucleotide probes in a particular set may be distinguished fromeither individual probes or other ligation products. The plurality ofoligonucleotide probe sets, the secondary extension products, and aligase are blended to form a ligase detection reaction mixture.

The ligase detection reaction mixture is subjected to one or more ligasedetection reaction cycles having a denaturation treatment andhybridization treatment substantially as described above. In thehybridization treatment, the oligonucleotide probe sets hybridize atadjacent positions in a base-specific manner to the respective secondaryextension products if present. As a result, adjacent probes ligate toone another to form a ligation product sequence containing thedetectable reporter label and the secondary extension product-specificportions connected together. The oligonucleotide probe sets mayhybridize to nucleotide sequences other than the respectivecomplementary secondary extension products but do not ligate togetherdue a presence of one or more mismatches and individually separateduring the denaturation treatment. Following the ligation detectionreaction cycles, the reporter labels of the ligation product sequencesare detected which indicates the presence of one or more targetnucleotide sequences in the sample.

FIGS. 1, 2, and 3 show flow diagrams of the primary PCR/secondaryPCR/LDR process of the present invention utilizing either of twodetection procedures. One alternative involves use of capillaryelectrophoresis or gel electrophoresis and a fluorescent quantificationprocedure. Alternatively, detection can be carried out by capture on anarray of capture oligonucleotide addresses and fluorescentquantification. FIG. 1 relates to detection of germline mutations (e.g.,a point mutation), while FIG. 2 detects biallelic polymorphisms, andFIG. 3 shows the detection of cancer-associated mutations.

FIG. 1 depicts the detection of a germline point mutation. In step 1,after DNA sample preparation, multiple exons are subjected to primaryPCR amplification using Taq (i.e. Thermus aquaticus) polymerase underhot start conditions with oligonucleotide primers having atarget-specific portion and a secondary primer-specific portion. At theend of the primary PCR phase, Taq polymerase may be inactivated byheating at 100° C. for 10 min or by a freeze/thaw step. The products ofthe primary PCR amplification phase are then subjected in step 2 tosecondary PCR amplification using Taq polymerase under hot startconditions with the secondary oligonucleotide primers. At the end of thesecondary PCR phase, Taq polymerase may be inactivated by heating at100° C. for 10 min or by a freeze/thaw step. In step 3, products of thesecondary PCR phase are then diluted 20-fold into fresh LDR buffercontaining LDR oligonucleotide probes containing allele-specificportions and common portions. Step 4 involves the LDR phase of theprocess which is initiated by addition of Taq ligase under hot startconditions. During LDR, oligonucleotide probes ligate to their adjacentoligonucleotide probes only in the presence of target sequence whichgives perfect complementarity at the junction site. The products may bedetected in two different formats. In the first format 5a,fluorescently-labeled LDR probes contain different length poly A orhexaethylene oxide tails. Thus, each ligation product sequence(resulting from ligation of two probes hybridized on normal DNA) willhave a slightly different length and mobility such that several ligationproduct sequences yield a ladder of peaks. Thus, each ligation product(resulting from ligation of two probes hybridized on normal DNA) willhave a slightly different length and mobility such that several ligationproduct sequences yield a ladder of peaks. A germline mutation wouldgenerate a new peak on the electropherogram. Alternatively, the LDRprobes may be designed such that the germline mutation ligation productsequence migrates with the same mobility as a normal DNA ligationproduct sequence, but it is distinguished by a different fluorescentreporter. The size of the new peak will approximate the amount of themutation present in the original sample; 0% for homozygous normal, 50%for heterozygous carrier, or 100% for homozygous mutant. In the secondformat 5b, each allele-specific probe contains e.g., 24 additionalnucleotide bases on their 5′ ends. These sequences are uniqueaddressable sequences which will specifically hybridize to theircomplementary address sequences on an addressable array. In the LDRreaction, each allele-specific probe can ligate to its adjacentfluorescently labeled common probe in the presence of the correspondingtarget sequence. Ligation product sequences corresponding to wild typeand mutant alleles are captured on adjacent addresses on the array.Unreacted probes are washed away. The black dots indicate 100% signalfor the wild type allele. The white dots indicate 0% signal for themutant alleles. The shaded dots indicate the one position of germlinemutation, 50% signal for each allele.

FIG. 2 depicts the detection of biallelic polymorphisms. In step 1,after DNA sample preparation, multiple exons are subjected to primaryPCR amplification using Taq polymerase under hot start conditions witholigonucleotide primers having a target-specific portion and a secondaryprimer-specific portion. The products of the primary PCR amplificationphase are then subjected in step 2 to secondary PCR amplification usingTaq polymerase under hot start conditions with the secondaryoligonucleotide primers. At the end of the secondary PCR phase, Taqpolymerase may be inactivated by heating at 100° C. for 10 min or by afreeze/thaw step. In step 3, products of the secondary PCR phase arethen diluted 20-fold into fresh LDR buffer containing LDRoligonucleotide probes containing allele-specific portions and commonportions. Step 4 involves the LDR phase of the process which isinitiated by addition of Taq ligase under hot start conditions. DuringLDR, oligonucleotide probes ligate to their adjacent oligonucleotideprobes only in the presence of target sequence which gives perfectcomplementarity at the junction site. In the first format 5a,fluorescently-labeled LDR probes contain different length poly A orhexaethylene oxide tails. Each ligation product sequence will have aslightly different length and mobility, such that several LDR productsyield a ladder of peaks. Alternatively, the LDR probes may be designedsuch that the ligation products for polymorphic alleles migrate at thesame position but are distinguished by different fluorescent reportergroups. The size of the peaks will approximate the amount of eachallele. In the second format 5b, each oligonucleotide probe containsunique addressable sequences with e.g., 24 additional nucleotide baseson their 5′ ends. These sequences will specifically hybridize to theircomplementary address sequences on an array of capture oligonucleotides.In the LDR phase, each allele-specific probe can ligate to its adjacentfluorescently labeled common probe in the presence of correspondingtarget sequence. Ligation product sequences corresponding to each alleleare captured on the array, while unligated oligonucleotide probes arewashed away. The black dots indicate that both chromosomes have a givenallele, the white dots show that neither chromosome has that allele, andthe shaded dots indicate that one chromosome has a given allele.

FIG. 3 depicts the detection of cancer-associated mutation. In step 1,after DNA sample preparation, multiple exons are subjected to primaryPCR amplification using Taq polymerase under hot start conditions witholigonucleotide primers having a target-specific portion and a secondaryprimer-specific portion. The products of the primary PCR amplificationphase are then subjected in step 2 to secondary PCR amplification usingTaq polymerase under hot start conditions with the secondaryoligonucleotide primers. At the end of the secondary PCR phase, Taqpolymerase may be inactivated by heating at 100° C. for 10 min or by afreeze/thaw step. Fluorescent quantification of PCR products can beachieved using capillary or gel electrophoresis in step 3. In step 4,the products are spiked with a 1/100 dilution of marker DNA (for each ofthe fragments). This DNA is homologous to wild type DNA, except itcontains a mutation which is not observed in cancer cells, but which maybe readily detected with the appropriate LDR probes. In step 5, themixed DNA products in products of the secondary PCR phase are thendiluted 20-fold into fresh LDR buffer containing LDR oligonucleotideprobes containing allele-specific portions and common portions. Step 6involves the LDR phase of the process which is initiated by addition ofTaq ligase under hot start conditions. During LDR, oligonucleotideprobes ligate to their adjacent oligonucleotide probes only in thepresence of target sequence which gives perfect complementarity at thejunction site.

The products may be detected in the same two formats discussed above. Inthe format of step 7a, products are separated by capillary or gelelectrophoresis, and fluorescent signals are quantified. Ratios ofmutant peaks to marker peaks give the approximate amount of cancermutations present in the original sample divided by 100. In the formatof step 7b, products are detected by specific hybridization tocomplementary sequences on an addressable array. Ratios of fluorescentsignals in mutant dots to marker dots give the approximate amount ofcancer mutations present in the original sample divided by 100.

As shown in FIG. 4, two DNA fragments of interest are treated with theprimary PCR/secondary PCR/LDR process of the present invention.Initially, the double stranded DNA molecules are denatured to separatethe strands. This is achieved by heating to a temperature of 80-105° C.Low concentrations of primary PCR oligonucleotide primers, containing a3′ target-specific portion (shaded area) and 5′ secondaryprimer-specific portion (black area), are then added and allowed tohybridize to the strands, typically at a temperature of 50-85° C. Athermostable polymerase (e.g., Taq aquaticus polymerase) is also added,and the temperature is then adjusted to 50-85° C. to extend the primeralong the length of the nucleic acid to which the primer is hybridized.After the extension phase of the polymerase chain reaction, theresulting double stranded molecule is heated to a temperature of 80-105°C. to denature the molecule and to separate the strands. Thesehybridization, extension, and denaturation steps may be repeated anumber of times to amplify the target to an appropriate level.

In the secondary PCR phase, the products of the primary PCR phase areblended with secondary PCR oligonucleotide primers and allowed tohybridize to one another, typically at a temperature of 50-85° C. Thesecondary oligonucleotide primers are usually used in higherconcentrations than are the primary oligonucleotide primers. Taqpolymerase is also added, and the temperature is then adjusted to 50-85°C. to extend the primer along the length of the primary PCR extensionproducts to which the secondary oligonucleotide primer is hybridized.After the extension phase of the polymerase chain reaction, theresulting double stranded molecule is heated to a temperature of 80-105°C. to denature the molecule and to separate the strands. Thesehybridization, extension, and denaturation steps may be repeated anumber of times to amplify the target to an appropriate level.

Once the secondary PCR phase of the process is completed, the ligationdetection reaction phase begins, as shown in FIG. 4. After denaturationof the target nucleic acid, if present as a double stranded DNAmolecule, at a temperature of 80-105° C., preferably 94° C., ligationdetection reaction oligonucleotide probes for one strand of the targetnucleotide sequence are added along with a ligase (for example, as shownin FIG. 4, a thermostable ligase like Thermus aquaticus ligase). Theoligonucleotide probes are then allowed to hybridize to the targetnucleic acid molecule and ligate together, typically, at a temperatureof 45-85° C., preferably, 65° C. When there is perfect complementarityat the ligation junction, the oligonucleotides can be ligated together.Where the variable nucleotide is T or A, the presence of T in the targetnucleotide sequence will cause the oligonucleotide probe with the F1reporter label to ligate to the common oligonucleotide probe with the 5′poly A tail A_(n), and the presence of A in the target nucleotidesequence will cause the oligonucleotide probe with the F2 reporter labelto ligate to the common oligonucleotide probe with A_(n). Similarly,where the variable nucleotide is A or G, the presence of T in the targetnucleotide sequence will cause the oligonucleotide probe with F3AAreporter label (i.e. the F3 reporter label coupled to 2 additional basesforming a 5′ poly A spacer) to ligate to the common oligonucleotideprobe with the 5′ poly A tail A_(n+4), and the presence of C in thetarget nucleotide sequence will cause the oligonucleotide probe with theF3 reporter label to ligate to the common oligonucleotide probe with the5′ poly A tail A_(n+4). Following ligation, the material is againsubjected to denaturation to separate the hybridized strands. Thehybridization/ligation and denaturation steps can be carried out throughone or more cycles (e.g., 1 to 50 cycles) to amplify target signals.Equimolar ligation of both F3-labeled oligonucleotides indicates theindividual is heterozygous for that locus, whereas ligation of only theF2 labeled oligonucleotides indicates the individual is homozygous forthe other locus.

In FIG. 4, the poly A_(n) and poly A_(n+4) tails are used wherequantification is to be carried out by capillary or gel electrophoresis.The tails of differing length cause the corresponding different ligationproduct sequences to form bands at different locations in the gel orcapillary. The presence of these bands at different locations permit thecorresponding nucleotide differences in the DNA being analyzed to beidentified. Although ligation product sequences can be distinguishedbased on the use of different reporter labels, the combination ofdifferent reporter labels and different length tails permits greaternumbers of nucleotide differences to be distinguished. This is importantfor multiplex detection processes.

As noted above with respect to FIGS. 1 to 3, detection can be carriedout on an addressable array instead of with gel or capillaryelectrophoresis. The use of such addressable arrays require that thepoly A tails on the LDR oligonucleotide probe not containing a reporterlabel (i.e. tails A_(n) and A_(n+4)) be replaced with differentaddressable array-specific oligonucleotide portions. As explained morefully infra, a solid support is provided with an array of captureoligonucleotides, some of which are complementary to the differentaddressable array-specific oligonucleotide portions. Hybridization ofthese portions to their complementary capture oligonucleotide probesindicates the presence of a corresponding nucleotide difference.

FIG. 5 is a schematic diagram of a primary PCR/secondary PCR/LDRprocess, in accordance with the present invention, where any possiblebase in 2 DNA molecules of interest are distinguished. The primary andsecondary PCR processes are carried out in substantially the same way asdescribed for FIG. 4. Appearance of fluorescent reporter labels F1, F2,F3, and F4 in conjunction with the left hand DNA molecule indicates thepresence of the A, G, C, and T alleles in the DNA molecule,respectively. As shown in FIG. 5, equal amounts of the F1 and F3reporter labels indicates that the individual in question isheterozygous for the A and C alleles. With respect to analysis of theright hand DNA molecule in FIG. 5, the same reporter label is used toindicate the presence of the different alleles; however, on eacholigonucleotide probe with the distinguishing bases, there are different5′ poly A tails. More particularly, a 2 unit poly A tail, a 4 unit polyA tail, a 6 unit poly A tail, and an 8 unit poly A tail correspond tothe T, C, G, and A alleles in the DNA molecule, respectively. As shownin FIG. 5, equal amounts of the F1 reporter label with the A₆ and A₄tails indicates that the individual in question is heterozygous for theG and C alleles.

FIG. 6 is a schematic diagram of a primary PCR/secondary PCR/LDRprocess, in accordance with the present invention, for detecting thepresence of any possible base at two nearby sites in DNA molecules ofinterest. The primary and secondary PCR phases are carried out insubstantially the same way as described for FIG. 4. Here, the LDR probesare able to overlap, yet are still capable of ligating provided there isperfect complementarity at the junction. This distinguishes LDR fromother approaches, such as allele-specific PCR where overlapping primerswould interfere with one another. In FIG. 6, the discriminatingoligonucleotide probes contain the reporter label with thediscriminating base on the 3′ end of these probes. The poly A tails areon the 3′ end of common oligonucleotide probes. In the left hand DNAmolecule, the presence of equal amounts of ligation product sequenceswith reporter labels F1 and F3 shows that the individual in question isheterozygous for the A and C alleles in the first position. Similarly,in the second position for the left hand DNA molecule, the presence ofligation product sequences with reporter labels F2, F3, and F4 showsthat the individual in question is heterozygous for the G, C, and Talleles. Turning to the right hand DNA molecule, the presence of equalamounts of ligation product sequences with reporter label F1 having theA₆ and A₄ tails indicates that at the first position, the individual inquestion is heterozygous for the G and C alleles. In the second positionfor the right hand DNA molecule, the presence of the equal amounts ofligation product sequences with reporter label F1 having the A₈ and A₂tails indicates that the individual in question is heterozygous for theA and T alleles.

FIG. 7 is a schematic diagram depicting the use of the primaryPCR/secondary PCR/LDR process of the present invention to detect a lowabundance mutation in the presence of an excess of normal sequence.Here, in the left hand DNA molecule is codon 12 of the K-ras gene,sequence GGT, which codes for glycine (“Gly”). A small percentage of thecells contain the G to A mutation in GAT, which codes for aspartic acid(“Asp”). The LDR probes for wild-type (i.e. normal) sequences aremissing from the reaction. If the normal LDR probes (with thediscriminating base being G) were included, they would ligate to thecommon probes and overwhelm any signal coming from the mutant target.Instead, as shown in FIG. 7, the existence of a ligation productsequence with fluorescent label F1 and the A_(n+2) tail indicates thepresence of the aspartic acid encoding mutant. In the right hand DNAmolecule, FIG. 7 shows codon 61 of the K-ras gene sequence CAG whichcodes for glutamine (“Gln”). A small percentage of the cells contain theC to G mutation in GAG, which codes for glutamic acid (“Glu”). Again,the LDR oligonucleotide probes do not include the C and A bases found inthe wild type form to avoid overwhelming the mutant signal. For this DNAmolecule, the existence of a ligation product sequence with fluorescentlabel F2 and the A_(n+4) tail indicates the presence of the glutamicacid encoding mutant.

II. LDR/PCR Process

A second aspect of the present invention relates to a method foridentifying one or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. This method has a ligasedetection reaction phase followed by a polymerase chain reaction phase.This method involves providing a sample potentially containing one ormore target nucleotide sequences with a plurality of sequencedifferences.

In the ligase detection reaction phase, one or more of oligonucleotideprobe sets are provided. Each set has a first oligonucleotide probe,having a target-specific portion and a 5′ upstream primer-specificportion, and a second oligonucleotide probe, having a target-specificportion and a 3′ downstream primer-specific portion. The oligonucleotideprobes in a particular set are suitable for ligation together whenhybridized adjacent to one another on a corresponding target nucleotidesequence. However, there is a mismatch which interferes with suchligation when they are hybridized to any other nucleotide sequencepresent in the sample. The sample, the plurality of oligonucleotideprobe sets, and a ligase are blended together to form a ligase detectionreaction mixture.

The ligase detection reaction mixture is subjected to one or more ligasedetection reaction cycles. These cycles include a denaturation treatmentand a hybridization treatment. In the denaturation treatment, anyhybridized oligonucleotides are separated from the target nucleotidesequences. The hybridization treatment causes the oligonucleotide probesets to hybridize at adjacent positions in a base-specific manner totheir respective target nucleotide sequences if present in the sample.Once hybridized, the oligonucleotide probe sets ligate to one another toform a ligation product sequence. This product contains the 5′ upstreamprimer-specific portion, the target-specific portions connectedtogether, and the 3′ downstream primer-specific portion. The ligationproduct sequence for each set is distinguishable from other nucleicacids in the ligase detection reaction mixture. The oligonucleotideprobe sets hybridized to nucleotide sequences in the sample other thantheir respective target nucleotide sequences but do not ligate togetherdue to a presence of one or more mismatches and individually separateduring the denaturation treatment.

In the polymerase chain reaction, one or a plurality of oligonucleotideprimer sets are provided. Each set has an upstream primer containing thesame sequence as the 5′ upstream primer-specific portion of the ligationproduct sequence and a downstream primer complementary to the 3′downstream primer-specific portion of the ligation product sequence,where one primer has a detectable reporter label. The ligase detectionreaction mixture is blended with the one or a plurality ofoligonucleotide primer sets and the polymerase to form a polymerasechain reaction mixture.

The polymerase chain reaction mixture is subjected to one or morepolymerase chain reaction cycles which include a denaturation treatment,a hybridization treatment, and an extension treatment. During thedenaturation treatment, hybridized nucleic acid sequences are separated.The hybridization treatment causes primers to hybridize to theircomplementary primer-specific portions of the ligation product sequence.During the extension treatment, hybridized primers are extended to formextension products complementary to the sequences to which the primersare hybridized. In a first cycle of the polymerase chain reaction phase,the downstream primer hybridizes to the 3′ downstream primer-specificportion of the ligation product sequence and is extended to form anextension product complementary to the ligation product sequence. Insubsequent cycles, the upstream primer hybridizes to the 5′ upstreamprimer-specific portion of the extension product complementary to theligation product sequence and the 3′ downstream primer hybridizes to the3′ downstream portion of the ligation product sequence.

Following the polymerase chain reaction phase of this process, thereporter labels are detected and the extension products aredistinguished to indicate the presence of one or more target nucleotidesequences in the sample.

FIG. 8 is a flow diagram depicting the LDR/PCR process of the presentinvention with or without restriction endonuclease digestion and usingcapillary electrophoresis detection. In step 1, a DNA sample is mixedwith Taq ligase and oligonucleotide probes containing a target-specificportion and a primer-specific portion. The mixture is subjected to anLDR process to produce ligation product sequences containing the ligatedtarget-specific portions and the primer-specific portions. Step 2involves mixing the ligation product sequences with Taq polymerase andprimers and subjecting the mixture to a PCR process. The next step isdetermined as a function of whether the ligation product sequences arethe same or different sizes. Where the ligation product sequences aredifferent sizes, step 3a is selected which involves subjecting theextension products from PCR to capillary electrophoresis or gelelectrophoresis, either of which is followed by fluorescentquantification. Step 3b is utilized where the ligation product sequencesare the same size and involves subjecting the extension products fromthe PCR phase to restriction endonuclease digestion. This generatesdigestion fragments of unique size which can be subjected to capillaryelectrophoresis or gel electrophoresis, followed by fluorescentquantification, according to step 4b. When step 3a is selected, thecurve generated as a result of electrophoresis shows three ligationproduct sequences migrating at lengths of 104, 107, and 110, with thepeak areas representing amplification of the Her-2 gene, loss ofheterozygosity of the p53 gene, and the control SOD gene, respectively.The electrophoresis curve where steps 3b and 4b are used involves threeligation product sequence restriction fragments at lengths of 58, 70,and 76, with the peak areas representing amplification of the Her-2gene, loss of heterozygosity of the p53 gene, and the control SOD gene,respectively.

As an alternative to FIG. 8, FIG. 9 shows the LDR/PCR process of thepresent invention where, in step 3, the extension products are capturedon an array of capture oligonucleotide addresses. The captureoligonucleotide probes can be complementary to a nucleotide sequenceacross the ligation junction. The number of gene copies captured on thearray of capture oligonucleotides is then determined by fluorescentquantification as compared with known controls. In FIG. 8, such analysisof the array indicates ligation product sequences hybridizing togene-specific addresses, where the fluorescent intensity representsamplification of the Her-2 gene, loss of heterozygosity of the p53 gene,and the control SOD gene, respectively.

FIG. 10 is a schematic diagram depicting an LDR/PCR process formultiplex detection of gene amplifications and deletions. Here, theratio of the Her-2/neu gene from Chromosome 17q, the p53 gene fromChromosome 17p, and the SOD gene from Chromosome 21q is detected.Following denaturation of DNA at 94° C., pairs of oligonucleotideprobes, having a target-specific portion and a primer-specific portion,are allowed to anneal adjacent to each other on target nucleic acids andligate to one another (in the absence of mismatches). This ligasedetection reaction is carried out with Tth ligase at ahybridization/ligation temperature of 65° C. which is well below theT_(m) values of 75° C. for the oligonucleotide probes. Next, theligation product sequences are simultaneously amplified by PCR using Taqpolymerase and two common primers complementary to the primer-specificportion, one of which is fluorescently labeled. This maintains theproportionality of the target sequences initially present in the sample.The extension products are then digested with HaeIII and HinpII whichreleases fluorescently labeled fragments of unique sizes for each targetsequence present in the sample. The digestion products are separated andanalyzed on an Applied Biosystems, Inc. (Foster City, Calif.) 373A DNASequencer. The peak heights and areas are related to the relative copiesof genes present in the initial target sample.

FIG. 11 is a schematic diagram, depicting a problem which can beencountered with the allele-specific LDR/PCR process. While a PCR/LDRprocess is very powerful, there may be circumstances where a multiplexedallele-specific LDR/PCR process of the present invention would bepreferred. The concept is to have one or more sets of LDRoligonucleotide probes, each set characterized by (a) a firstoligonucleotide probe, having a target-specific portion and a 5′upstream primer-specific portion, and (b) a second oligonucleotideprobe, having a target-specific portion and a 3′ downstreamprimer-specific portion. As shown in step 1 of FIG. 11, the LDRoligonucleotide probes anneal adjacent to each other on the targetsequence. An LDR reaction using thermostable ligase (black dot) wouldform a ligation product sequence provided there is perfectcomplementarity at the ligation junction. In step 2, the ligationproduct sequences are PCR amplified with primer sets, each setcharacterized by (a) an upstream primer containing the same sequence asthe 5′ upstream primer-specific portion of a ligation product sequenceand (b) a downstream primer complementary to the 3′ downstreamprimer-specific portion of that ligation product sequence. The primersare shown as black lines in step 2. If one primer is fluorescentlylabeled, it will generate a fluorescent product which may be detected ina variety of detection schemes. For the LDR/PCR process to be specific,a PCR extension product should not be formed in the absence of aligation event. Unfortunately, the possibility exists for polymerase toextend the first LDR oligonucleotide probe (off normal target), forminga product containing the length of the target sequence, and aprimer-specific portion on the 5′ end. Meanwhile, polymerase can makeseveral complementary copies of the downstream LDR probe using thedownstream primer. In a second amplification cycle, this downstream LDRprobe extension product can anneal to the upstream LDR probe extensionproduct off the target sequence, and generate a sequence containing thetarget region flanked by the two primer-specific sequences. This productwill amplify as the LDR product, and thus yield a false positive signal.

FIG. 12 is a schematic drawing showing a solution to the allele specificLDR/PCR problem, utilizing an intermediate exonuclease digestion step.Allele-specific LDR/PCR can be achieved while significantly reducingbackground ligation independent (incorrect) target amplification. To doso, it is necessary to eliminate one or more of the components requiredfor ligation independent PCR amplification, without removing theinformation content of the ligation product sequence. One solution is touse exonuclease in step 2 to digest unreacted LDR oligonucleotide probesfrom step 1. By blocking the end which is not ligated, for example the3′ end of the downstream oligonucleotide probe, one probe can be madesubstantially resistant to digestion, while the other is sensitive. Onlythe presence of full length ligation product sequence will preventdigestion of the upstream primer. Blocking groups include use of athiophosphate group and/or use of 2-O-methyl ribose sugar groups in thebackbone. Exonucleases include Exo I (3′-5′), Exo III (3′-5′), and ExoIV (both 5′-3′ and 3′-5′), the later requiring blocking on both sides.One convenient way to block both probes is by using one long “padlock”probe (see M. Nilsson et. al., “Padlock Probes: CircularizingOligonucleotides for Localized DNA Detection,” Science 265: 2085-88(1994), which is hereby incorporated by reference), although this is byno means required. An advantage of using exonucleases, for example acombination of Exo I (single strand specific) and Exo III (double strandspecific), is the ability to destroy both target and one LDR probe,while leaving the ligation product sequences substantially undigested.By using an exonuclease treatment prior to PCR, in accordance with steps3 and 4, either one or both oligonucleotide probes in each set aresubstantially reduced, and thus hybridization of the remainingoligonucleotide probes to the original target DNA (which is alsosubstantially reduced by exonuclease treatment) and formation of aligation product sequence which is a suitable substrate for PCRamplification by the oligonucleotide primer set is substantiallyreduced. In other words, formation of ligation independent labeledextension products is substantially reduced or eliminated.

FIG. 13 is a flow diagram showing an allele-specific LDR/PCR processusing exonuclease digestion with either size based- or DNA arraybased-detection. The flow diagram shows the three reactions required forthe multiplexed allele-specific LDR/PCR process. In step 1, sets of LDRoligonucleotide probes (wherein the downstream probes are blocked ontheir 3′ ends) are ligated in the presence of the correct allele targetusing Taq DNA ligase. Unreacted upstream probes are digested withexonuclease in step 2, coincidentally, target is also digested. Finally,in step 3, primer sets are used to amplify the ligation product sequenceby hybridizing to the primer specific portions of ligation productsequences. In step 4a, the LDR oligonucleotide probes in a givenparticular set generate a unique length product, and thus may bedistinguished from either oligonucleotide probes or other ligationproducts. After the PCR reaction, the products are separated by size orelectrophoretic mobility. Labels on the PCR primers are detected, andthe products are distinguished by size. In step 4b, the LDRoligonucleotide probes in a particular set use may be distinguished fromeither oligonucleotide probes or other ligation product sequences bydifferences in the sequences of the PCR primers. By using a plurality ofoligonucleotide primer sets, each set characterized by (a) an upstreamprimer containing the same sequence as the 5′ upstream primer-specificportion of a ligation product sequence, and (b) a downstream primercomplementary to the 3′ downstream primer-specific portion of thatligation product sequence, wherein one primer has a detectable reporterlabel and the other primer contains an addressable nucleotide sequencelinked to the 5′ end of that primer such that the addressable nucleotidesequence remains single stranded after a PCR reaction, all the productscan be distinguished. The latter may be achieved by using a non-naturalbase within a PCR primer which polymerase cannot extend through, thusgenerating PCR products which have single stranded tails. See C. Newton,et. al., “The Production of PCR Products with 5′ Single-stranded TailsUsing Primers that Incorporate Novel Phosphoramidite Intermediates,”Nucl. Acids Res. 21(3): 1155-62 (1993), which is hereby incorporated byreference. By providing a DNA array with different captureoligonucleotides immobilized at different particular sites, where thecapture oligonucleotides have nucleotide sequences complementary to theaddressable nucleotide sequences on the primers, the PCR extensionproducts can hybridize to the DNA array. Finally, the labels ofextension product sequences captured using the addressablearray-specific portions immobilized to the DNA array at particular sitescan be detected. This indicates the presence of one or more targetnucleotide sequences in the sample.

FIG. 14 is a flow diagram showing a quantitative allele-specific LDR/PCRprocess using exonuclease digestion in step 3 with either size based- orDNA array based-detection. The flow diagram shows how one can quantifythe amounts of different targets (especially low abundance cancermutations) by adding marker sequence(s) (step 1) at the start of the LDRreaction (step 2). In this embodiment, the biochemical reactions (i.e.PCR (step 4)) are followed, as described in FIG. 13, and the relativeamount of mutant product to marker product are quantified usingcapillary or gel electrophoresis (step 5a) or capture on an addressablearray (step 5b). The amount of mutant target present in the originalsample can then be determined.

FIG. 15 is a schematic drawing showing an allele-specific LDR/PCRprocess with exonuclease digestion (step 2) for detection of mutationsor polymorphisms. Mutations and polymorphisms may be distinguished asdescribed in FIG. 12. In this example, in step 1, the upstream LDRoligonucleotide probes, which have the discriminating allele-specificbase at the 3′ end of the target-specific portion, have different 5′upstream primer-specific portions. Thus, different primers (in the PCRamplification step (i.e. step 3)) may be labeled with differentfluorescent groups (Fam and Tet) to allow for distinction of products(step 4). An array based detection scheme may also be used, where theupstream (allele-specific) probes have different 5′ upstreamprimer-specific portions, and the different PCR primers containdifferent addressable nucleotide sequences which remain single strandedafter a PCR reaction.

FIG. 16 is a schematic drawing showing an allele-specific LDR (step1)/PCR (step 3) process using exonuclease digestion (step 2) fordetection of mononucleotide or dinucleotide repeat polymorphisms. One ofthe most powerful uses of LDR/PCR is for detecting nucleotide repeatpolymorphisms, a task which cannot be achieved by allele-specific PCR(because the 3′ nucleotide is always the same), nor easily achieved byobserving PCR product size variation (due to Taq polymerase slippageduring amplification). In FIG. 16, the LDR oligonucleotide probesdistinguish between an A₉ and A₁₀ mononucleotide repeat sequence byvirtue of the specificity of thermostable DNA ligase. LDR products areonly formed on the correct length target sequence, and thus the presenceof that target is distinguished (step 4).

FIG. 17 is schematic drawing showing an allele-specific LDR/PCR processusing exonuclease digestion (step 2) for detection of low abundancemononucleotide or dinucleotide repeat mutations. Mononucleotide repeatlength mutations may be distinguished as described in FIG. 12. In FIG.17, the LDR oligonucleotide probes (step 1) distinguish between an A₈,A₉ (mutants), and A₁₀ (normal) mononucleotide repeat sequences by virtueof the specificity of thermostable DNA ligase. The two upstream LDRoligonucleotide probes differ in the length of the mononucleotidesequence at their 3′ ends of their target specific portion and havedifferent 5′ upstream primer-specific portions. Thus, different primers(in the PCR amplification step (step 3)) may be labeled with differentfluorescent groups (Fam and Tet) to allow for distinction of products(step 4). This has the distinct advantage of allowing mononucleotiderepeat polymorphisms to be distinguished based on fluorescent labelinstead of size, the latter being susceptible to false positives due topolymerase slippage. An array based detection scheme may also be used,where the upstream (allele-specific) probes have different 5′ upstreamprimer-specific portions, and the different PCR primers containdifferent addressable nucleotide sequences which remain single strandedafter a PCR reaction.

FIG. 18 is a flow diagram, showing an allele-specific LDR/PCR processusing uracil N-glycosylase selection with either size based- or DNAarray based-detection. The flow diagram shows the four reactionsrequired for multiplexed allele-specific LDR/PCR. Sets of LDRoligonucleotide probes (wherein one or both probes contain deoxy-uracilin place of deoxythimidine) are ligated in the presence of the correctallele target using Taq DNA ligase in step 1. A complementary copy ofthe ligation product sequence is made with sequenase in step 2.Sequenase is a modified T7 polymerase, with any easily inactivatedpolymerase (i.e. mesophilic polymerases such as, E. coli polymerase)being useful. Both ligation product sequences and unreacted probes aredestroyed with uracil N-glycosylase in step 3. The advantage of usinguracil N-glycosylase is its proven ability in carry-over prevention forPCR. Finally PCR primer sets are used to amplify the sequenase extensionproducts in step 4. In step 5a, the LDR oligonucleotide probes in aparticular set generate a unique length product, and thus may bedistinguished from either probes or other ligation products. After thePCR reaction, the products are separated by size or electrophoreticmobility. Labels on the PCR primers are detected, and products aredistinguished by size. In step 5b, the LDR oligonucleotide probes in aparticular set may be distinguished from either probes or other ligationproduct sequences by differences in the sequences of the primer-specificportions. By using a plurality of oligonucleotide primer sets, each setcharacterized by (a) an upstream primer containing the same sequence asthe 5′ upstream primer-specific portion of a ligation product sequence,and (b) a downstream primer complementary to the 3′ downstreamprimer-specific portion of that ligation product sequence. One primerhas a detectable reporter label, and the other primer contains anaddressable array-specific portion linked to the 5′ end of that primersuch that the addressable array-specific portion remains single strandedafter a PCR reaction, one can distinguish all the products. By providinga DNA array with different capture oligonucleotides immobilized atdifferent particular sites, wherein the capture oligonucleotides havenucleotide sequences complementary to the addressable array-specificportions on the primers, the PCR extension products can be hybridized tothe DNA array. Finally, the labels of extension product sequencescaptured using the addressable nucleotide sequence portions andimmobilized to the DNA array at particular sites can be detected toindicate the presence of one or more target nucleotide sequences in thesample.

FIG. 19 is a flow diagram showing a quantitative allele-specific LDR/PCRprocess using uracil N-glycosylase selection with either size based- orDNA array based-detection. The flow diagram shows how one can quantifythe amounts of different targets (especially low abundance cancermutations) by adding marker sequence(s) in step 1 at the start of theLDR phase in step 2. As described in FIG. 18, the biochemical reactions(i.e. sequenase treatment (step 3), uracil N-glycosylase selection (step4), and PCR (step 5)) are preceded with, and the relative amount ofmutant product to marker product is quantified using capillary or gelelectrophoresis (step 6a) or capture on an addressable array (step 6b).From this information, the amount of mutant target present in theoriginal sample can be determined.

FIG. 20 is a schematic drawing showing an allele-specific LDR/PCRprocess using uracil N-glycosylase selection (step 3) (after sequenasetreatment (step 2)) for detection of mononucleotide or dinucleotiderepeat polymorphisms. One of the most powerful uses of the LDR/PCRprocess is for detecting nucleotide repeat polymorphisms, a task whichcannot be achieved by allele-specific PCR (since the 3′ nucleotide isalways the same), nor easily achieved by observing PCR product sizevariation (due to Taq polymerase slippage during amplification), as instep 4. In FIG. 20, the LDR (step 1) oligonucleotide probes distinguishbetween an A₉ and A₁₀ mononucleotide repeat sequence by virtue of thespecificity of thermostable DNA ligase. Ligation product sequences areonly formed on the correct length target sequence, and, thus, thepresence of that target is distinguished in step 5.

FIG. 21 is a schematic drawing showing an allele-specific LDR/PCRprocess using uracil N-glycosylase selection for detection of lowabundance mononucleotide or dinucleotide repeat mutations.Mononucleotide repeat length mutations may be distinguished as describedin FIG. 18. In FIG. 21, the LDR oligonucleotide probes distinguishbetween an A₈, A₉ (mutants), and A₁₀ (normal) mononucleotide repeatsequences by virtue of the specificity of thermostable DNA ligase (step1). Sequenase treatment (step 2) and uracil N-glycosylase selection(step 3) are then carried out. The two upstream LDR oligonucleotideprobes differ in the length of the mononucleotide sequence at the 3′ends of their target-specific portion, and have different 5′ upstreamprimer-specific portions. Thus, different primers (in the PCRamplification step (steps 4-5)) may be labeled with differentfluorescent groups (Fam and Tet) to allow for distinction of products.This has the distinct advantage of allowing one to distinguishmononucleotide repeat polymorphisms based on fluorescent label insteadof size, the latter being susceptible to false positives due topolymerase slippage. An array based detection scheme may also be used,where the upstream (allele-specific) probes have different 5′ upstreamprimer-specific portions, and the different PCR primers containdifferent addressable array-specific portions which remain singlestranded after a PCR reaction.

The LDR/exonuclease/PCR process described with reference to FIGS. 11 to17 and the LDR/sequenase/uracil N-glycosylase/PCR process set forth inFIGS. 18-21 provide the ability to multiplex detect and then PCR amplifymany different target sequences and to distinguish multiple single-baseor sequence variations, all in a single reaction tube. This is achievedby combining the sensitivity of PCR with the selectivity of LDR. Sincethe selection of mutant sequences is mediated by LDR rather than PCR,the primary PCR/secondary PCR/LDR process is less susceptible tofalse-positive signal generation. In addition, the primary PCR/secondaryPCR/LDR process allows detection of closely-clustered mutations,detection of single base or small insertions and deletions in smallrepeat sequences, quantitative detection of less than 1% mutations inhigh background of normal DNA, and detection of ligation productsequences using addressable arrays. Detection of single base or smallinsertions and deletions in small and medium repeat sequences may cause“stutter” when the primary amplification is PCR. No othercurrently-available technique can adequately solve this problem,especially when the target sequence containing the mononucleotide repeatpolymorphism is present in a lower abundance than normal DNA. In fact,analysis of genomic mutations which involve repeat sequence changes isseverely hampered by the PCR “stutter” problem. By using the LDR/PCRprocess of the present invention, it is possible to detect down to 1%mutations in a high background of normal DNA. The only relatively minorchallenges presented by this process are that the mutations must beknown and that 3 different enzymes/reaction conditions must be utilized.

III. Primary PCR/Secondary PCR Process

A third aspect of the present invention also involves a method foridentifying two or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations inone or more target nucleotide sequences. This method involves subjectinga sample potentially containing one or more target nucleotide sequenceswith a plurality of sequence differences to two successive polymerasechain reaction phases.

For the first polymerase chain reaction phase, one or more primaryoligonucleotide primer groups are provided where each group comprisesone or more primary oligonucleotide primer sets. Each set has a firstoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primary-specific portion, and a secondoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primer-specific portion. The first oligonucleotideprimers of each set in the same group contain the same 5′ upstreamsecondary primer-specific portion and the second oligonucleotide primersof each set in the same group contain the same 5′ upstream secondaryprimer-specific portion. The oligonucleotide primers in a particular setare suitable for hybridization on complementary strands of acorresponding target nucleotide sequence to permit formation of apolymerase chain reaction product. However, there is a mismatch whichinterferes with formation of such a polymerase chain reaction productwhen the primary oligonucleotide primers hybridize to any othernucleotide sequence present in the sample. The polymerase chain reactionproducts in a particular set may be distinguished from other polymerasechain reaction products with the same group or other groups. The primaryoligonucleotide primers are blended with the sample and the polymeraseto form a primary polymerase chain reaction mixture.

The primary polymerase chain reaction mixture is subjected to two ormore polymerase chain reaction cycles involving a denaturationtreatment, a hybridization treatment, and an extension treatment, asdescribed above. During the hybridization treatment, the target-specificportion of a primary oligonucleotide primer is hybridized to the targetnucleotide sequences. In the extension treatment, the hybridized primaryoligonucleotide primers are extended to form primary extension productscomplementary to the target nucleotide sequence to which the primaryoligonucleotide primer is hybridized.

Although the upstream secondary primer-specific portions of a primaryoligonucleotide primer set are not present on the target DNA, theirsequences are copied by the second and subsequent cycles of the primarypolymerase chain reaction phase. As a result, the primary extensionproducts produced after the second cycle have the secondaryprimer-specific portions on their 5′ ends and the complement ofprimer-specific portion on their 3′ ends.

In the second polymerase chain reaction phase of this aspect of thepresent invention, one or a plurality of secondary oligonucleotideprimer sets are provided. Each set has a first secondary primer having adetectable reporter label and containing the same sequence as the 5′upstream portion of a first primary oligonucleotide primer, and a secondsecondary primer containing the same sequence as the 5′ upstream primerof the second primary oligonucleotide primer from the same primaryoligonucleotide primer set as the first primary oligonucleotidecomplementary to the first secondary primer. A set of secondaryoligonucleotide primers amplify the primary extension products in agiven group. The secondary oligonucleotide primers are blended with theprimary extension products and the polymerase to form a secondarypolymerase chain reaction mixture.

The secondary polymerase chain reaction mixture is subjected to one ormore polymerase chain reaction cycles involving a denaturationtreatment, a hybridization treatment, and an extension treatment, asdescribed above. In the hybridization treatment, the secondaryoligonucleotide primers are hybridized to the primary extensionproducts, while the extension treatment causes the hybridized secondaryoligonucleotide primers to be extended to form secondary extensionproducts complementary to the primary extension products. Aftersubjecting the secondary polymerase chain reaction mixture to the two ormore polymerase chain reaction cycles, the labelled secondary extensionproducts are detected. This indicates the presence of one or more targetnucleotide sequences in the sample.

FIG. 22 is a flow diagram depicting a primary PCR/secondary PCR process,in accordance with the present invention, for detection ofmicrosatellite repeats. In step 1 (i.e. the primary PCR phase), afterDNA sample preparation, multiple exons are amplified using Taqpolymerase under hot start conditions with oligonucleotide primershaving a target-specific portion and a secondary primer-specificportion. Step 2 involves a secondary PCR phase where Taq polymerase isused to amplify the primary PCR extension products with oligonucleotideprimers containing the same sequence as the secondary primer-specificportion of the primary PCR primers. The extension products resultingfrom the secondary PCR phase are subjected in step 3 to capillaryelectrophoresis or gel electrophoresis, followed by fluorescentquantification. The electrophoresis results in FIG. 22 indicate thepresence of both alleles (i.e., chromosomes) containing RB1 and NM23 andloss of heterozygosity (i.e., loss of allele on one chromosome) for p53.

FIG. 23 is a schematic diagram depicting a primary PCR/secondary PCRprocess, according to the present invention, for detection of the lossof heterozygosity due to insertions and deletions in microsatelliterepeats. The primary PCR phase in step 1 is initiated by denaturing thesample DNA at 94° C. Long PCR oligonucleotide primers, having 3′ endscomplementary to unique DNA surrounding microsatellite repeat sequencesand 5′ ends containing the same sequence as one of two primers utilizedin the secondary PCR phase, are then caused to anneal to target DNA at65° C. The primary PCR phase is carried out for 10-15 cycles. The longprimers utilized in the primary PCR phase can be multiplexed as long asthey do not amplify alleles with overlapping length ranges. Thesereactions must be carried out on tumor and corresponding normal DNA toidentify informative (i.e. heterozygous) loci. In step 2 (i.e. secondaryPCR amplification), primers complementary to the 5′ ends of the primaryPCR primers (one fluorescently labeled) are then used to amplify theprimary PCR extension products at nearly equal efficiency. The secondaryPCR extension products are then separated and analyzed by gelelectrophoresis and an Applied Biosystems Inc. 373A DNA Sequencer usingthe Genescan 672 software package. Areas of loss of heterozygosity atinformative loci are identified. The analysis in FIG. 23 shows thepresence of both alleles (i.e., chromosomes) containing RB1 and NM23 andloss of heterozygosity (i.e., loss of allele on one chromosome) for p53.

IV. General Process Information

The ligase detection reaction is described generally in WO 90/17239 toBarany et al., F. Barany et al., “Cloning, Overexpression and NucleotideSequence of a Thermostable DNA Ligase-encoding Gene,” Gene, 109:1-11(1991), and F. Barany, “Genetic Disease Detection and DNA AmplificationUsing Cloned Thermostable Ligase,” Proc. Natl. Acad. Sci. USA,88:189-193 (1991), the disclosures of which are hereby incorporated byreference. In accordance with the present invention, the ligasedetection reaction can use 2 sets of complementary oligonucleotides.This is known as the ligase chain reaction which is described in the 3immediately preceding references, which are hereby incorporated byreference. Alternatively, the ligase detection reaction can involve asingle cycle which is known as the oligonucleotide ligation assay. SeeLandegren, et al., “A Ligase-Mediated Gene Detection Technique,” Science241:1077-80 (1988); Landegren, et al., “DNA Diagnostics—MolecularTechniques and Automation,” Science 242:229-37 (1988); and U.S. Pat. No.4,988,617 to Landegren, et al., which are hereby incorporated byreference

During ligase detection reaction phases, the denaturation treatment iscarried out at a temperature of 80-105° C., while hybridization takesplace at 50-85° C. Each cycle comprises a denaturation treatment and athermal hybridization treatment which in total is from about one to fiveminutes long. Typically, the ligation detection reaction involvesrepeatedly denaturing and hybridizing for 2 to 50 cycles. The total timefor the ligase detection reaction phase is 1 to 250 minutes.

The oligonucleotide probe sets or primers can be in the form ofribonucleotides, deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, modified phosphate-sugar-backboneoligonucleotides, nucleotide analogs, and mixtures thereof.

In one variation, the oligonucleotides of the oligonucleotide probe setseach have a hybridization or melting temperature (i.e. T_(m)) of 66-70°C. These oligonucleotides are 20-28 nucleotides long.

The oligonucleotide probe sets or primers, as noted above, have areporter label suitable for detection. Useful labels includechromophores, fluorescent moieties, enzymes, antigens, heavy metals,magnetic probes, dyes, phosphorescent groups, radioactive materials,chemiluminescent moieties, and electrochemical detecting moieties.

The polymerase chain reaction process is fully described in H. Erlich,et. al., “Recent Advances in the Polymerase Chain Reaction,” Science252: 1643-50 (1991); M. Innis, et. al., PCR Protocols: A Guide toMethods and Applications, Academic Press: New York (1990); and R. Saiki,et. al., “Primer-directed Enzymatic Amplification of DNA with aThermostable DNA Polymerase,” Science 239: 487-91 (1988), which arehereby incorporated by reference.

A particularly important aspect of the present invention is itscapability to quantify the amount of target nucleotide sequence in asample. This can be achieved in a number of ways by establishingstandards which can be internal (i.e. where the standard establishingmaterial is amplified and detected with the sample) or external (i.e.where the standard establishing material is not amplified, and isdetected with the sample).

In accordance with one quantification method, the signal generated bythe reporter label, resulting from capture of ligation product sequencesproduced from the sample being analyzed, are detected. The strength ofthis signal is compared to a calibration curve produced from signalsgenerated by capture of ligation product sequences in samples with knownamounts of target nucleotide sequence. As a result, the amount of targetnucleotide sequence in the sample being analyzed can be determined. Thistechniques involves use of an external standard.

Another quantification method, in accordance with the present invention,relates to an internal standard. Here, a known amount of one or moremarker target nucleotide sequences is added to the sample. In addition,a plurality of marker-specific oligonucleotide probe sets are addedalong with the ligase, the previously-discussed oligonucleotide probesets, and the sample to a mixture. The marker-specific oligonucleotideprobe sets have (1) a first oligonucleotide probe with a target-specificportion complementary to the marker target nucleotide sequence, and (2)a second oligonucleotide probe with a target-specific portioncomplementary to the marker target nucleotide sequence and a detectablereporter label. The oligonucleotide probes in a particularmarker-specific oligonucleotide set are suitable for ligation togetherwhen hybridized adjacent to one another on a corresponding marker targetnucleotide sequence. However, there is a mismatch which interferes withsuch ligation when hybridized to any other nucleotide sequence presentin the sample or added marker sequences. The presence of ligationproduct sequences is identified by detection of reporter labels. Theamount of target nucleotide sequences in the sample is then determinedby comparing the amount of ligation product sequence generated fromknown amounts of marker target nucleotide sequences with the amount ofother ligation product sequences.

Another quantification method, in accordance with the present inventioninvolves, analysis of a sample containing two or more of a plurality oftarget nucleotide sequences with a plurality of sequence differences.Here, ligation product sequences corresponding to the target nucleotidesequences are detected and distinguished by any of thepreviously-discussed techniques. The relative amounts of the targetnucleotide sequences in the sample are then quantified by comparing therelative amounts of captured ligation product sequences generated. Thisprovides a quantitative measure of the relative level of the targetnucleotide sequences in the sample.

The preferred thermostable ligase is that derived from Thermusaquaticus. This enzyme can be isolated from that organism. M. Takahashi,et al., “Thermophillic DNA Ligase,” J. Biol. Chem. 259:10041-47 (1984),which is hereby incorporated by reference. Alternatively, it can beprepared recombinantly. Procedures for such isolation as well as therecombinant production of Thermus aquaticus ligase (as well as Thermusthemophilus ligase) are disclosed in WO 90/17239 to Barany, et. al., andF. Barany, et al., “Cloning, Overexpression and Nucleotide Sequence of aThermostable DNA-Ligase Encoding Gene,” Gene 109:1-11 (1991), which arehereby incorporated by reference. These references contain completesequence information for this ligase as well as the encoding DNA. Othersuitable ligases include E. coli ligase, T4 ligase, and Pyococcusligase.

The ligation detection reaction mixture may include a carrier DNA, suchas salmon sperm DNA.

The hybridization step in the ligase detection reaction, which ispreferably a thermal hybridization treatment discriminates betweennucleotide sequences based on a distinguishing nucleotide at theligation junctions. The difference between the target nucleotidesequences can be, for example, a single nucleic acid base difference, anucleic acid deletion, a nucleic acid insertion, or rearrangement. Suchsequence differences involving more than one base can also be detected.Preferably, the oligonucleotide probe sets have substantially the samelength so that they hybridize to target nucleotide sequences atsubstantially similar hybridization conditions. As a result, the processof the present invention is able to detect infectious diseases, geneticdiseases, and cancer. It is also useful in environmental monitoring,forensics, and food science.

A wide variety of infectious diseases can be detected by the process ofthe present invention. Typically, these are caused by bacterial, viral,parasite, and fungal infectious agents. The resistance of variousinfectious agents to drugs can also be determined using the presentinvention.

Bacterial infectious agents which can be detected by the presentinvention include Escherichia coli, Salmonella, Shigella, Klebsiella,Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis,Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella,Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcusaureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria,Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea,Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis,Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponemapalladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsialpathogens, Nocardia, and Acitnomycetes.

Fungal infectious agents which can be detected by the present inventioninclude Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasmacapsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candidaalbicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrixschenckii, Chromomycosis, and Maduromycosis.

Viral infectious agents which can be detected by the present inventioninclude human immunodeficiency virus, human T-cell lymphocytotrophicvirus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis CVirus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses,orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses,rhabdo viruses, polio viruses, toga viruses, bunya viruses, arenaviruses, rubella viruses, and reo viruses.

Parasitic agents which can be detected by the present invention includePlasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodiumovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosomaspp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonasspp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobiusvermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculusmedinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystiscarinii, and Necator americanis.

The present invention is also useful for detection of drug resistance byinfectious agents. For example, vancomycin-resistant Enterococcusfaecium, methicillin-resistant Staphylococcus aureus,penicillin-resistant Streptococcus pneumoniae, multi-drug resistantMycobacterium tuberculosis, and AZT-resistant human immunodeficiencyvirus can all be identified with the present invention.

Genetic diseases can also be detected by the process of the presentinvention. This can be carried out by prenatal or post-natal screeningfor chromosomal and genetic aberrations or for genetic diseases.Examples of detectable genetic diseases include: 21 hydroxylasedeficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome,Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heartdisease, single gene diseases, HLA typing, phenylketonuria, sickle cellanemia, Tay-Sachs Disease, thalassemia, Klinefelter Syndrome, HuntingtonDisease, autoimmune diseases, lipidosis, obesity defects, hemophilia,inborn errors of metabolism, and diabetes.

Cancers which can be detected by the process of the present inventiongenerally involve oncogenes, tumor suppressor genes, or genes involvedin DNA amplification, replication, recombination, or repair. Examples ofthese include: BRCA1 gene, p53 gene, APC gene, Her2/Neu amplification,Bcr/Abl, K-ras gene, and human papillomavirus Types 16 and 18. Variousaspects of the present invention can be used to identify amplifications,large deletions as well as point mutations and smalldeletions/insertions of the above genes in the following common humancancers: leukemia, colon cancer, breast cancer, lung cancer, prostatecancer, brain tumors, central nervous system tumors, bladder tumors,melanomas, liver cancer, osteosarcoma and other bone cancers, testicularand ovarian carcinomas, head and neck tumors, and cervical neoplasms.

In the area of environmental monitoring, the present invention can beused for detection, identification, and monitoring of pathogenic andindigenous microorganisms in natural and engineered ecosystems andmicrocosms such as in municipal waste water purification systems andwater reservoirs or in polluted areas undergoing bioremediation. It isalso possible to detect plasmids containing genes that can metabolizexenobiotics, to monitor specific target microorganisms in populationdynamic studies, or either to detect, identify, or monitor geneticallymodified microorganisms in the environment and in industrial plants.

The present invention can also be used in a variety of forensic areas,including for human identification for military personnel and criminalinvestigation, paternity testing and family relation analysis, HLAcompatibility typing, and screening blood, sperm, or transplantationorgans for contamination.

In the food and feed industry, the present invention has a wide varietyof applications. For example, it can be used for identification andcharacterization of production organisms such as yeast for production ofbeer, wine, cheese, yogurt, bread, etc. Another area of use is withregard to quality control and certification of products and processes(e.g., livestock, pasteurization, and meat processing) for contaminants.Other uses include the characterization of plants, bulbs, and seeds forbreeding purposes, identification of the presence of plant-specificpathogens, and detection and identification of veterinary infections.

Desirably, the oligonucleotide probes are suitable for ligation togetherat a ligation junction when hybridized adjacent to one another on acorresponding target nucleotide sequence due to perfect complementarityat the ligation junction. However, when the oligonucleotide probes inthe set are hybridized to any other nucleotide sequence present in thesample, there is a mismatch at a base at the ligation junction whichinterferes with ligation. Most preferably, the mismatch is at the baseadjacent the 3′ base at the ligation junction. Alternatively, themismatch can be at the bases adjacent to bases at the ligation junction.

As noted supra, detection and quantification can be carried out usingcapillary or gel electrophoresis or on a solid support with an arraycapture oligonucleotides.

The use of capillary and gel electrophoresis for such purposes is wellknown. See e.g., Grossman, et. al., “High-density Multiplex Detection ofNucleic Acid Sequences Oligonucleotide Ligation Assay and Sequence-codedSeparation,” Nucl. Acids Res. 22(21): 4527-34 (1994), which is herebyincorporated by reference.

The use of a solid support with an array of capture oligonucleotides isfully disclosed in pending provisional U.S. Patent Application Ser. No.60/011,359, which is hereby incorporated by reference. When using sucharrays, the oligonucleotide primers or probes used in theabove-described coupled PCR and LDR phases, respectively, have anaddressable array-specific portion. After the LDR or PCR phases arecompleted, the addressable array-specific portions for the products ofsuch processes remain single stranded and are caused to hybridize to thecapture oligonucleotides during a capture phase. C. Newton, et al., “TheProduction of PCR Products With 5′ Single-Stranded Tails Using PrimersThat Incorporate Novel Phosphoramidite Intermediates,” Nucl. Acids Res.21(5):1155-62 (1993), which is hereby incorporated by reference.

During the capture phase of the process, the mixture is contacted withthe solid support at a temperature of 45-90° C. and for a time period ofup to 60 minutes. Hybridizations may be accelerated by adding cations,volume exclusion or chaotropic agents. When an array consists of dozensto hundreds of addresses, it is important that the correct ligationproduct sequences have an opportunity to hybridize to the appropriateaddress. This may be achieved by the thermal motion of oligonucleotidesat the high temperatures used, by mechanical movement of the fluid incontact with the array surface, or by moving the oligonucleotides acrossthe array by electric fields. After hybridization, the array is washedsequentially with a low stringency wash buffer and then a highstringency wash buffer.

It is important to select capture oligonucleotides and addressablenucleotide sequences which will hybridize in a stable fashion. Thisrequires that the oligonucleotide sets and the capture oligonucleotidesbe configured so that the oligonucleotide sets hybridize to the targetnucleotide sequences at a temperature less than that which the captureoligonucleotides hybridize to the addressable array-specific portions.Unless the oligonucleotides are designed in this fashion, false positivesignals may result due to capture of adjacent unreacted oligonucleotidesfrom the same oligonucleotide set which are hybridized to the target.

The capture oligonucleotides can be in the form of ribonucleotides,deoxyribonucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, peptide nucleotide analogues, modified peptidenucleotide analogues, modified phosphate-sugar backboneoligonucleotides, nucleotide analogues, and mixtures thereof.

Where an array is utilized, the detection phase of the process involvesscanning and identifying if LDR or PCR products have been produced andcorrelating the presence of such products to a presence or absence ofthe target nucleotide sequence in the test sample. Scanning can becarried out by scanning electron microscopy, confocal microscopy,charge-coupled device, scanning tunneling electron microscopy, infraredmicroscopy, atomic force microscopy, electrical conductance, andfluorescent or phosphor imaging. Correlating is carried out with acomputer.

EXAMPLES LDR/PCR Process Example 1 Genomic DNA Preparation

Genomic DNA was prepared from the blood of two normal human volunteers,one male and one female, according to standard techniques. Briefly,approximately 12 ml of blood was obtained in EDTA-containing bloodcollection tubes. Red blood cells were lysed by mixing the blood sampleswith 4 volumes of lysis buffer (10 mM Tris pH 8.0, 10 mM EDTA). After 10min on ice with occasional agitation, the suspensions were centrifugedand the supernatants were decanted. The white blood cell pellets wereresuspended in 20 ml of lysis buffer, and the above process wasrepeated. Each cell pellet was then suspended in 15 ml of digestionbuffer (50 mM Tris pH 8.0, 5 mM EDTA, 100 mM NaCl, 1% SDS) and 3 mg (0.2mg/ml) of proteinase K was added. The cells were digested at 37° C. for5 hours. The digests were extracted twice with equal volumes of phenol,then once with equal volumes of a 1:1 phenol:chloroform mixture andfinally once with equal volumes of chloroform, each time centrifugingthe mixture and removing the aqueous phase for the next extraction.After the final extraction and removing the aqueous phases, one tenthvolume of 3 M sodium acetate, pH 6.5, was added. Two volumes of ice cold100% EtOH were then added to each solution to precipitate the genomicDNAs, which were spooled out of solution on glass pipettes. The DNAprecipitates were washed twice in 0.75 ml volumes of 70% EtOH, brieflycentrifuging each time to allow removal of the supernatants. Afterremoving the supernatants for the second time, the remaining EtOH wasallowed to evaporate and the DNA was suspended in 0.5 ml of TE (10 mMTri-HCl pH 8.0 containing 1 mM EDTA) solution. A fifth dilution of eachDNA solution was also prepared in TE.

To determine the concentrations of the one fifth DNA solutions, 1, 2,and 4 μl aliquots of each were loaded on a 1% agarose gel with a knownamount of HindIII digested lambda DNA as a control. The gel was run at150 Volts for 2 hours with ethidium bromide in the electrophoresisbuffer. After photographing the gel and comparing the intensities of theDNA bands, the one fifth dilutions were judged to have concentrations ofapproximately 100 ng/ml. DNA solutions extracted from various tumor celllines were the generous gifts of other laboratories. The concentrationsof these solutions were checked in a similar fashion and solutions of100 ng/ml in TE were prepared.

To digest the genomic DNAs with Taq I, 25 μl of the 100 ng/μl solutionswas mixed with 5 μl of 10× medium salt buffer (0.5 M NaCl, 0.1 M MgCl₂,0.1 M Tris, pH 8.0), 20 μl of water-ME (i.e. water containing 6 mM ME(i.e., mercaptoethanol)), and 400 U of Taq I restriction endonuclease.The digests were covered with mineral oil and incubated at 65° C. for 1hour. The reactions were stopped by adding 1.2 μl of 500 mM EDTA andheating the specimens to 85° C. for 10 min. Complete digestion of theDNAs was checked by electrophoresing aliquots on a 1% agarose gel.

Example 2 Oligonucleotide Preparation for LDR Probes and PCR Primers

All oligonucleotides were synthesized on a 394A DNA Synthesizer (AppliedBiosystems Division of Perkin-Elmer Corp., Foster City, Calif.).Oligonucleotides labeled with 6-FAM were synthesized using themanufacturer's suggested modifications to the synthesis cycle (AppliedBiosystems Inc., 1994) and were subsequently deprotected at 55° C. for 4hr. LDR oligonucleotides were purified by ethanol precipitation afterovernight deprotection at 55° C. The primer-specific portions of theoligonucleotides used for PCR amplification were purified bypolyacrylamide gel electrophoresis on 10% acrylamide/7M urea gels.Oligonucleotides were visualized after electrophoresis by UV shadowingagainst a lightening screen and excised from the gel (Applied BiosystemsInc., 1992). They were then eluted overnight at 64° C. in TNE (i.e.Tris-sodium EDTA) buffer (100 mM Tris/HCl pH 8.0 containing 500 mM NaCland 5 mM EDTA) and recovered from the eluate using Sep Pak cartridges(Millipore Corp, Milford, Mass.) following the manufacture'sinstructions.

Oligonucleotides were resuspended in 100 μl TE (i.e. 10 mM Tri-HCl pH8.0 containing 1 mM EDTA). Typical concentrations of these original LDRprobe solutions are about 1 μg/μl or approximately 74 pm/μl asdetermined by the following formula:

[concentration (μg/μl)×10⁶]/[length(nt)×325]=pm/μl

The concentrations of the LDR probes are given in Table 1. Theconcentrations of oligonucleotides complementary to the oligonucleotideprobes of the ligase detection reaction were higher. ZipALg1F was 3.75μg/μ1 and ZipBLg2R was 2.01 μg/μl or 524 pm/μl and 281 pm/μl,respectively, as determined by the formula above.

TABLE 1 Primer Length μg/μl pm/μl Vol = 100pm l = 200pm G6PDEx6-3L 48 nt0.86 55.1 1.81 μl 3.63 μl G6PDEx6-4R 48 0.65 41.7  2.4  4.8 ErbBEx1-5L48 0.95 60.9 1.64 3.28 ErbBEx1-5R 48 1.4025 89.9 1.11 2.22 Int2Ex3-7L 501.6005 98.5 1.02 2.03 Int2Ex3-8R 46 1.306 87.4 1.14 2.29 p53Ex8-9L  21.036 61.3 1.63 3.26 p53Ex8-10R 44 1.164 81.4 1.23 2.46 SODEx3-11L 491.287 80.8 1.24 2.48 SODEx3-12R 47 1.2045 78.9 1.27 2.53

As a prerequisite for the LDR phase, the downstream LDR oligonucleotidesprobes were phosphorylated with T4 polynucleotide kinase. Aliquots ofthe 5 downstream oligonucleotides equivalent to 200 pm (see Table 1)were combined with 10 μl of 10× kinase buffer (500 mM Tris/HCl pH 8.0,100 mM MgCl₂), 10 μl of 10 mM ATP, 20 U T4 kinase, and sufficientwater-ME to give a final volume of 100 μA Phosphorylation was carriedout at 37° C. for 30 min followed by incubation for 10 min at 85° C. toinactivate the T4 enzyme. The resulting concentration of the kinased LDRprobe solution was 2 pm/μl or 2000 fm/μl in each probe.

The kinase reaction is summarized as follows:

4.8 μl G6PDEx6-4R 2.2 μl ErbBEx1-5R 2.3 μl Int2Ex3-8R 2.5 μl p53Ex8-10R2.5 μl SODEx3-12R 10 μl 10 x Kinase Buffer 10 μl 10 mMATP 65.7 μl HOH +ME 100 μl Total +2 μl = 20 units T4 Kinase 37° C. for 30 min. Heat killkinase, 85° C., 10 min. Final concentration = 2 pm/μl = 2000 fm/μl

The solutions of the LDR and PCR oligonucleotides were adjusted toconvenient concentrations. The kinased LDR probe solution was dilutedfourfold in water to yield a concentration of 500 fm/μl. A solution ofthe upstream LDR probes was made by combining volumes of the probesequivalent to 200 pm (see Table 1) with sufficient water to give a finalvolume of 400 μl. This created a solution 500 fm/μl in each of theupstream LDR probes. Aliquots (20 μl) of the kinased and unkinased LDRprobes were frozen for subsequent use. Standard solutions of the PCRprimers (10 pm/μl) were prepared from their original solutions bycombining 9.5 μl of ZipALg1F and 17.8 μl of ZipBLg2R with sufficientwater to achieve a total volume of 500 μl. These solutions were frozenfor use in the LDR/PCR process.

Unkinased probes were prepared according to the following:

200 pm ea Primer 3.62 μl G6PDEx6-3L 3.28 μl ErbBEx1-5L 2.04 μlInt2Ex3-7L 3.26 μl p53Ex8-9L 2.48 μl SODEx3-11L 385.32 μl HOH 400 μlTotal Vol Final concentration = 0.5 pm/μl = 500 fm/μl

TABLE 2 Sequences Probe (length in nt) Gene Location Upstream DownstreamLigation Position erb 17q12-q21 erbBEx1-5L (48) erbBEx1-6R (48) exon “1”P40 G6PD Xq28 G6PDEx6-3L (48) G6PDEx6-4R (48) exon 6 W1145 Int2 11q13Int2Ex3-7L (50) Int2Ex3-8R (46) exon 3 W135 p53 17p13.1 p53Ex8-9L (52)p53Ex8-10R (44) exon 8 P51 SOD 21q22.1 SODEx3-11L (49) SODEx3-12R (47)exon 3 P355

FIG. 24 shows the design of LDR oligonucleotide probes forquantification of gene amplifications and deletions in the LDR/PCRprocess. These oligonucleotide probes were designed to recognize exon 8in the p53 tumor suppressor gene (on chromosome 17p), exon 3 of int-2(on chromosome 11q), an internal exon in HER-2/neu (i.e. HER-2/neu/erbBoncogene) (on chromosome 17q), exon 3 in SOD (i.e. super oxide dimutase)(on chromosome 21q), and exon 6 in G6PD (i.e. glucose 6-phosphatedehydrogenase) (on chromosome Xq). Each pair of LDR oligonucleotideprobes has the following features: (i) The left oligonucleotide probecontains from 5′ to 3′ an 18 base sequence identical to thefluorescently labeled secondary oligonucleotide primer (black bar), an“adjustment sequence” (white bar), and a target-specific sequence offrom 22 to 28 bases with a T_(m) of 75° C. (patterned bar); (ii) Theright oligonucleotide probe contains from 5′ to 3′ a target-specificsequence of 20-25 bases with a T_(m) of 75° C. (patterned bar), a singleHaeIII or HinP1I restriction site at slightly different positions withinthe target-specific sequence, and an “adjustment sequence” (white bars).The two oligonucleotide probes are designed such that their combinedlength is exactly 96 bases, with 50 G+C bases and 46 A+T bases. Theposition of each unique restriction site generates a product whichdiffers by at least 2 bases from the other products. Eacholigonucleotide probe set has an exon-specific region chosen to ligatethe junction sequence of (A, T)C C(A, T). This junction sequencecorresponds to either a proline residue (codon CCN) or the complementarysequence of a tryptophan residue (TGG). These sequences were chosen tominimize differences in ligation rates and the chance of a polymorphismat the ligation junction.

LDR Probe Sequences

G6PDEx6-3L (SEQ. ID. NO. 1)5′CAC GCT ATC CCG TTA GAC ATT GTC AAG CAG GCG ATG TTG TCC CGG TTC 3′G6PDEx6-4R (SEQ. ID. NO. 2)5′CAG ATG GGG CCG AAG ATC CTG TTA TTG ATA CAT AGT GCG GTA GTT GGC 3′erbBEx1-5L (SEQ. ID. NO. 3)5′CAC GCT ATC CCG TTA GAC ATC GCC CTG ATG GGG AGA ATG TGA AAA TTC 3′erbBEx1-6R (SEQ. ID. NO. 4)5′CAG TGG CCA TCA AAG TGT TGA GGG AGC GTA CAT AGT GCG GTA GTT GGC 3′Int2Ex3-7L (SEQ. ID. NO. 5)5′CAC GCT ATC CCG TTA GAC ATT CAT AAC CCT TGC CGT TCA CAG ACA CGT AC 3′Int2Ex3-8R (SEQ. ID. NO. 6)5′CAC AGT CTC TCG GCG CTG GGC AAT AAT ACA TAG TGC GGT AGT TGG C 3′p53Ex8-9L (SEQ. ID. NO. 7)5′CAC GCT ATC CCG TTA GAC ATC TTA GTA ATT GAG GTGCGT GTT TGT GCC TGT C 3′ p53Ex8-10R (SEQ. ID. NO. 8)5′CTG GGA GAG ACC GGC GCA CAT TAC TAC ATA GTG CGG TAG TTG GC 3′SODEx-3-11L (SEQ. ID. NO. 9)5′CAC GCT ATC CCG TTA GAC ATC TGT ACC AGT GCA GGT CCT CAC TTT AAT C 3′SODEx-3-12R (SEQ. ID. NO. 10)5′CTC TAT CCA GAA AAC ACG GTG GGC CGC TAC ATA GTG CGG TAG TTG GC 3′

PCR Primers:

ZipALg1F (SEQ. ID. NO. 11) 5′ Fam-GGA GCA CGC TAT CCC GTT AGA C 3′ (Tm =71° C.) ZipBLg2R (SEQ. ID. NO. 12) 5′ CGC TGC CAA CTA CCG CAC TAT G 3′(Tm = 72° C.)(Underlined sequences are common between LDR probes and ZipALg1F or thecomplement of ZipBLg2R.)

Example 3 Buffers and Reagents

A. LDR Buffers/Reagents—the following LDR buffers and reagents wereselected:

-   10× ST ligase buffer (0.2 M Tris pH 8.5, 0.1 M MgCl₂) [This was also    tested with Tris at pH 7.6.]-   10× TT ligase buffer (0.2 M Tris pH 7.6, 0.5 M KCl, 0.1 M MgCl₂, 5    mM EDTA)-   NAD (10 mM)-   DTT (200 mM)-   LDR primer solution containing one tenth concentration of each of    the LDR primer mixtures (50 fm of each LDR primer per μl)-   Tth DNA Ligase (625 U/μl)    PCR Buffers/Reagents—the following PCR buffers and reagents were    selected:-   10× Stoffel buffer (0.1 M KCl, 0.1M Tris-HCl pH 8.3 Perkin Elmer)-   dNTP solution (100 mM total, 25 mM of each dNTP Perkin Elmer),    diluted 5 fold in dHOH to a final concentration of 5 mM of each dNTP-   ZipALg1F (10 pm/μl)-   ZipBLg2R (10 pm/μl)

Example 4 LDR/PCR Process

Four LDR/PCR processes were performed for each DNA to be tested withreaction tubes PCR amplified to 22, 24, 26, and 30 cycles to assure thatone reaction would be halted in the exponential phase. Each LDR reaction(20 μl) was thermal cycled and then a PCR mix (30 μl) containing primerswith a portion complementary to the primer-specific portion of the LDRprobes was added to each specimen to allow exponential amplification. Tominimize differences between reaction tubes, master mixes of LDR and PCRreagents were made.

A master mix of LDR reagents was constructed with a volume sufficientfor all reaction tubes. Proportions and volumes for a single reactionwere as follows:

Reagent Volume 10X ST Ligase Buffer 2 μl NAD (10 mM) 2 μl DTT (200 mM) 1μl dHOH_(—) 5 μl Total 10 μl Tth DNA Ligase 0.2 μl (=125 U)Mixes of target DNA and LDR probes for each reaction tube wereconstructed with the following proportions:

Reagent Volume DNA (TaqI digested)  1 μl (=50 ng) LDR Probe Mix  4 μl(200 fm each primer) dHOH_(—)  5 μl Total 10 μl

For each reaction, 10 μl was placed in a thin-walled PCR tube, rapidlymixed with 10 μl of LDR reagent mix (including ligase), overlayed withmineral oil, and placed in a Perkin Elmer 9600 thermal cycler.

LDR was initiated by holding at 96° C. for 2 minutes to denature the DNAfollowed by 10 cycles of 94° C. for 30 seconds and 65° C. for 4 minutes.

PCR reagent mixes for each reaction tube were constructed with thefollowing proportions:

Reagent Volume 10X Stoffel buffer 5 μl dNTP solution 8 μl (=0.8 mM eachdNTP in final reaction) ZipALg1F (10 pm/μl) 2.5 μl (=25 pm per reaction)ZipBLg2R (10 pm/μl) 2.5 μl (=25 pm per reaction) dHOH 12 μl Total 30 μlStoffel Fragment 0.25 μl (=2.5 U)

At the completion of the LDR reaction, the tubes were held at 94° C.,while 30 ml of PCR reagent mix (including Stoffel fragment) were addedto each tube. PCR amplification was accomplished by thermal cycling at94° C. for 15 seconds followed by 60° C. for 50 seconds. At 22, 24, 26,and 30 cycles, respectively, one of four identical reaction tubes ofeach DNA specimen was removed and quenched in a slurry of dry ice andETOH.

Example 5 Agarose Gel Evaluation

Ten microliter aliquots of the 26 and 30 cycle reaction specimens wereevaluated on a 2% agarose gel. Ethidium bromide staining revealed bandsof the expected size (104 bp).

Example 6 Digestion of Products, Preparation of Dilutions, and Loadingon GeneScanner

To separate the gene-specific LDR/PCR products, 10 μl aliquots of the22, 24, and 26 cycle reactions were digested by adding 10 μl of asolution containing 5 U each of HaeIII and HinP1I restriction enzymes(both from New England BioLabs), 2 μl of 10× restriction enzyme buffernumber 2 (New England BioLabs), and 8 μl of dHOH (i.e. distilled water).The digests were incubated at 37° C. for one hour and then stopped bythe addition of 1 μl of 0.5 M EDTA, pH 8.0. The restriction digests werea one half dilution of the original LDR/PCR products. A 10 fold dilutionof each sample was also prepared by adding 5 μl of each restrictiondigest to 20 μl of TE buffer.

Before loading samples on the ABI 373A DNA Sequencer (AppliedBiosystems) a 1:5 mixture of 50 mM EDTA pH 8.0 containing 2% bluedextran and de-ionized formamide was made. To 5 μl of the EDTA-BlueDextran solution, 5 μl of digested LDR/PCR product dilution and 1 μl ofGENESCAN 1000 ROX marker (Applied Biosystems) were added. Thesesolutions were heated to 85° C. for 10 minutes and snap chilled on ice,before 5.5 μl were loaded on the denaturing gel.

Samples were analyzed in an Applied Biosystems 373A DNA sequencer on a0.4 mm thick, 10% polyacrylamide/7M urea gel with a well-to-readdistance of 12 cm. The gel matrix was buffered with 1.2×TBE (106 mMTris-borate and 2.4 mM EDTA pH 8.3) and the electrophoresis chamberbuffer contained 0.6×TBE (53.4 mM Tris-borate and 1.2 mM EDTA pH 8.3).The gel was pre-run prior to sample loading at 1600 V for 30 minuteswith the electrode polarity reversed (anode in the chamber with samplewells at the top of the gel). After loading, the gene-specific LDR/PCRproducts were electrophoresed at 1200 V and the primary data wascaptured using the ABI 672 Data Collection Software V1.1 (AppliedBiosystems)

Using the ABI 672 GeneScan Analysis Software V1.2.2 (AppliedBiosystems), the resulting data were displayed as electropherograms,with peak heights and peak areas calculated.

In the normal female, the ErbB2 peak is lower, and the p53 peak isslightly lower than the remaining 3 peaks. See FIGS. 25A-D. In differentexperiments, it was observed that the ErbB2 peak is always lower, theG6PD, Int-2, p53, and SOD peak areas would vary somewhat, but all 5peaks would retain the same relative profile from one sample to the nextfor a given experiment. When comparing male with female DNA, the G6PDpeak was about half the area of other peaks, consistent with a singleX-chromosome in males, while the other peaks were essentially the same.The ErB2 peak for the NM10 Breast Cancer cell line is slightly elevated,while that in cell line SKBR3 is several fold greater than the normalfemale control, reflecting the known ErbB-2 gene amplification in thesetwo cell lines. In addition, cell line NM10 appears to have undergoneLOH (i.e. a loss of heterozygosity) of p53, while cell line SKBR3appears to have undergone LOH of G6PD and p53. Some of the cells in cellline SKBR3 may have lost both copies of the p53 gene. Repeating theseamplifications in the absence of the ErbB-2 primers was used to confirmthe presence of these additional gene deletions (see below).

These results can be quantified by comparing the ratio of peak areas ineach peak to a standard (the SOD peak area) for that experiment. The rawdata and ratio of peak areas are given below:

TABLE 3 Raw Peak Area Data Genes ErB G6PD Int2 p53 SOD Male 9954 2152545688 36346 62506 Female 8340 39309 39344 30270 54665 NM10 20096 5548367083 17364 84339 SKBR3 106650 19120 50103 2888 48119

TABLE 4 Ratio of Peak Areas to SOD Peak Area ErbB/SOD G6PD/SOD Int2/SODp53/SOD Male 0.16 0.34 0.73 0.58 Female 0.15 0.72 0.72 0.55 NM10 0.240.66 0.80 0.21 SKBR3 2.22 0.40 1.04 0.06

Although the ratios differ for each gene, (due to different efficienciesof LDR/PCR for each gene,) the ratios are generally consistent betweenthe male and female sample, except for the G6PD/SOD ratio. The G6PD forthe female is about twice the value as the male, accurately reflectingthe presence of two and one X chromosome, respectively. One can quantifythe amount of ErbB2 amplification by comparing the ratio of peak arearatios between normal DNA and cancer cell lines.

TABLE 5 Ratio of Peak Areas Ratios ErbB/2 G6PD Int2 p53 Female/Male 0.962.09 0.98 0.95 NM10/Male 1.50 1.91 1.09 0.35 SKBR3/Male 13.92 1.15 1.420.10

From these ratios, it can be determined that the normal male and femalehave the same number of genes on chromosomes 17q (ErbB), 17p (p53), and11q (Int 2), but that the female has twice as many G6PD genes, or Xchromosomes. Likewise, cell line NM10 showed slight amplification of theErbB-2 gene, and LOH at p53, while cell line SKBR3 shows significantamplification of the ErbB-2 gene, LOH at G6PD and p53. To confirmadditional gene amplifications and deletions, primer pairs causingmassive amplifications may be removed from the LDR/PCR reaction (seebelow).

In the normal female, the ErbB2 peak is lower than the remaining 4peaks. In different experiments, it was observed that the G6PD, Int-2,p53, and SOD peak areas would vary somewhat, but would retain the samerelative profile from one sample to the next. See FIGS. 26A-C. The ErbB2peak was consistently lower, and slight shoulders were observed on theG6PD and SOD peaks, for unknown reasons. The ErbB-2 peak in both cellline samples is several fold greater than the normal female control,reflecting the known ErbB-2 gene amplification in these two cell lines.In addition, the ZR-75-30 line appears to show LOH of p53, while theSKGT-2 cell line appears to have a slight amplification of the Int-2region. By repeating these LDR/PCR experiments in the absence of theErbB-2 primers, it was demonstrated that these results are not artifactsof the massive levels of ErbB-2 amplification. See FIGS. 27A-C. Bothgene amplifications and deletions for multiple genes using the LDR/PCRformat have been demonstrated. See FIGS. 26A-C and 27A-C.

Again, these results can be quantified by comparing the ratio of peakareas in each peak to a standard (the SOD peak area) for thatexperiment. The raw data and ratio of peak areas are given below:

TABLE 6 Raw Peak Area Data Genes ErbB G6PD Int2 p53 SOD Female; 4 PrimerSets NA 9577 8581 9139 8128 ZR7530; 4 Primer Sets NA 8452 7904 4168 7996SKGT2; 4 Primer Sets NA 15915 28614 13116 12478 Female; 5 Primer Sets3955 9436 8066 9304 8848 ZR7530; 5 Primer Sets 66748 11105 8812 41639303 SKGT2; 5 Primer Sets 263254 21877 31887 13630 13480

TABLE 7 Ratio of Peak Areas to SOD Peak Area ErbB/SOD G6PD/SOD Int2/SODp53/SOD Female; 4 Primer Sets NA 1.18 1.06 1.12 ZR7530; 4 Primer Sets NA1.06 0.99 0.52 SKGT2; 4 Primer Sets NA 1.28 2.29 1.05 Female; 5 PrimerSets 0.45 1.97 0.91 1.05 ZR7530; 5 Primer Sets 7.17 1.19 0.95 0.45SKGT2; 5 Primer Sets 19.53  1.62 2.37 1.01

The ratios are remarkably consistent between the four primer set and thefive primer set experiments. The only exception is the G6PD peak for theSKGT2 cell line, where the huge peak for ErbB-2 may have added to theG6PD peak.

One can quantify the amount of ErbB2 and Int-2 amplification as well asp53 deletion by comparing the ratio of peak area ratios between normalDNA and cancer cell lines, as shown in Table 8. In addition, the ratiosfrom using 4 sets of primers can be compared with 5 sets of primers toascertain the internal consistency of this technique.

TABLE 8 Ratio of Peak Area Ratios ErbB G6PD Int2 p53 Female; 4/5 NA 1.101.16 1.07 ZR7530; 4/5 NA 0.89 1.04 1.16 SKGT2; 4/5 NA 0.79 0.97 1.04ZR7530/Female; 4/4 NA 0.90 0.94 0.46 ZR7530; Female; 5/5 16.05 1.12 1.040.43 SKGT2/Female; 4/4 NA 1.08 2.17 0.93 SKGT2; Female; 5/5 43.69 1.522.59 0.96

The values for the top half of Table 8 should all be close to 1.0 if theLDR/PCR technique is internally consistent, when using 4 or 5 primers.All values are very close to 1.0. Again, the value for G6PD for SKGT2 isa bit low for the reasons mentioned.

The values on the bottom half of Table 8 show the extent of ErbB-2amplification. The numbers are quite consistent for the 4 primer and 5primer amplifications (with the exception of SKGT2-G6PD noted above).The ZR7530 cell line demonstrates a clear LOH for p53, while the SKGT2cell line shows amplification of the Int-2 region, and both p53 genespresent.

Primary PCR/Secondary PCR/LDR Process Example 7 OligonucleotideSynthesis

Oligonucleotides were assembled by standard phosphoramidite chemistry onan Expedite DNA synthesizer (Perseptive Biosystems, Framingham, Mass.).Oligonucleotides 5′-end labeled with 6-FAM, TET, and HEX weresynthesized using the appropriate dye phosphoramidites (PerkinElmer-Applied Biosystems) and purified with Oligonucleotide PurificationCartridges (Perkin Elmer-Applied Biosystems) following themanufacturer's protocol (Applied Biosystems Division-Perkin Elmer Corp.,“Synthesis and Purification of Fluorescently Labeled OligonucleotidesUsing Dye Phosphoramidites,” User Bulletin, number 78, AppliedBiosystems Division, Foster City, Calif., (1994)), which is herebyincorporated by reference. All oligonucleotides were checked for purityon an Applied Biosystems 270-HT capillary electrophoresis instrumentusing a mPAGE-3 column (J&W Scientific, Folsom, Calif.). Onlyoligonucleotides that were greater than 95% pure were used for theexperiments. Oligonucleotides were resuspended in 250 ml TE (10 mMTris/HCl and 5 mM EDTA pH 8.0). Typical concentrations were 300-500 mMfor crude stock solutions and 100-200 mM for OPC (i.e. OligonucleotidePurification Columns available from Applied Biosystems) purified stocksolutions. For PCR and LDR, oligonucleotides were diluted to workingsolutions of 10 mM (10 pmoles/ml) or 5 mM (5 pmoles/ml).

Example 8 Phosphorylation of LDR Oligonucleotides

The 12 LDR common oligonucleotides were phosphorylated at the 5′ end topermit ligation to the fluorescent labeled oligonucleotides. Theoligonucleotides are shown below in Table 9.

TABLE 9 LDR Oligonucleotide Sequences Allele-Specific OligonucleotideCommon Oligonucleotide Locus (5′-->3′) (5′-->3′)  1FAM-AGCTTCAATGATGAGAACCTGC P-GCATAGTGGTGGCTGACCTGTTCATATSEQ. ID. NO. 13) (SEQ. ID. NO. 14) TET-AGCTTCAATGATGAGAACCTGT(SEQ. ID. NO. 15)  2 FAM-CTCCATGGGCCCAGCC P-AGCACTGGTGCCCTGTGAG(SEQ. ID. NO. 16) (SEQ. ID. NO. 17) TET-CTCCATGGGCCCAGCT(SEQ. ID. NO. 18)  3 FAM-GGGGACAGCCATGCACTGA P-GCCTCTGGTAGCCTTTCAACCATA(SEQ. ID. NO. 19) (SEQ. ID. NO. 20) TET-GGGGACAGCCATGCACTGC(SEQ. ID. NO. 21)  4 FAMTTAGAAATCATCAAGCCTAGGTCATP-CACCTTTTAGCTTCCTGAGCAATGAT (SEQ. ID. NO. 22) (SEQ. ID. NO. 23)TET-TTAGAAATCATCAAGCCTAGGTCAG (SEQ. ID. NO. 24)  5HEX-GGTTGTATTTGTCACCATATTAATTA P-ATTTTTCTCTATTGTTTTCATCTTTCAGGA(SEQ. ID. NO. 25) (SEQ. ID. NO. 26) HEX-ATGGTTGTATTTGTCACCATATTAATTG(SEQ. ID. NO. 27)  6 FAM-GGGCCAAGAAGGTATCTACCAP-ATAGTGTCTATTAGGCATTTGAAAATGTGTAT (SEQ. ID. NO. 28) (SEQ. ID. NO. 29)TET-GGGCCAAGAAGGTATCTACCG (SEQ. ID. NO. 30)  7FAM-ACACAGCAGCTTACTCCAGAGG P-TCAAGTCCAAGGCCATTGGCTTATA (SEQ. ID. NO. 31)(SEQ. ID. NO. 32) TET-ACACAGCAGCTTACTCCAGAGA (SEQ. ID. NO. 33)  8FAM-CCAGCAAAGAGAAAAGAAGGG P-CCCCAGAAATCACAGGTGGGCTAT (SEQ. ID. NO. 34)(SEQ. ID. NO. 35) TET-CCAGCAAAGAGAAAAGAAGGA (SEQ. ID. NO. 36)  9FAM-ATGATATTAGAGCTCACTCATGTCCA P-TCAGTTTGGAAAAAGACAAAGAATTCTTT(SEQ. ID. NO. 37) (SEQ. ID. NO. 38) TET-ATGATATTAGAGCTCACTCATGTCCG(SEQ. ID. NO. 39) 10 HEX-TGCTGTCTTCCAGGAATCTGTTP-CAACTCTCTCGAAGCCATGTTCACAA (SEQ. ID. NO. 40) (SEQ. ID. NO. 41)HEX-ATTGCTGTCTTCCAGGAATCTGTG (SEQ. ID. NO. 42) 11HEX-GGACATAGTGACCGTGCAGGTC P-CTTCCCCAGTGTGAGTGCCGTA (SEQ. ID. NO. 43)(SEQ. ID. NO. 44) HEX-ATGGACATAGTGACCGTGCAGGTT (SEQ. ID. NO. 45) 12HEX-CTATGACACCGTCATCAGCAGG P-GACATCCAGGCCCCCGAC (SEQ. ID. NO. 46)(SEQ. ID. NO. 47) HEX-TACTATGACACCGTCATCAGCAGA (SEQ. ID. NO. 48) Theallele-specific oligonucleotides are 5′ end labeled with either FAM,TET, or HEX. All the common oligonucleotides are phosphorylated at the5′ end. Underline denotes tails that are not complementary to the targetsequence. LDR primer sets were designed in two ways: (i) allele-specificprimers were of the same length but contained either FAM or TET label;or (ii) the allele-specific primers were both labeled with HEX butdiffered in length by two bases.

This was accomplished either during the synthesis with Phosphate-ON(Clontech Laboratories, Palo Alto, Calif.) according to themanufacturer's instructions or post-synthesis, using T4 polynucleotidekinase (Boehringer Mannheim, Indianapolis, Ind.). In the latter, acommon oligomer was diluted into 50 μl of kinase buffer (50 mM Tris/HCl,10 mM MgCl₂, and 1 mM ATP) to a final concentration of 1 mM (500 pmol in50 μl). Ten units of T4 kinase was added, and the reaction was incubatedat 37° C. for 30 min. The T4 kinase was inactivated by heating at 95° C.for 10 min.

The kinase reaction was carried out as follows:

-   -   10 μl 50 mM common oligo    -   5 μl 10× kinase buffer    -   5 μl 10 mM ATP    -   30 μl H2O    -   50 μl Total

+1 μl T4 Kinase (10 units)

-   -   37° C. for 30 min.    -   95° C. for 10 min.    -   Final concentration=10 mM

Example 9 Multiplex PCR Amplification

Twelve gene regions (2-13) were chosen for simultaneous PCRamplification based on information available in the Human GenomeDatabase, as shown below in Table 10.

TABLE 10 List of Polymorphic Sites Analyzed Site Locus Chr. NucleotideNumber Symbol Locus Name Location Position Variation Het. Ref. 1 CYP2D6cytochrome P450IID6 22q13.1 4469 (M33388) C, T .38 2 2 AT3 antithrombinIII 1q23-q25.1 7987 (X68793) C, T .46 3 3 C6 complement 5p14-p12 185(X72179) A, C .47 4 component C6 4 IL1A interleukin 1 alpha 2q13 6282(X03833) T, G .34 5 5 NF1 neurofibromatosis 17q11.2 63683 A, G .47 6(L05367) 6 ALDOB aldolase B 9q22.3-q31 1087 (M15656) A, G .50 7 7 A2Malpha 2 12p13.3-p12.3 153 (X68731) G, A .42 8 macroglobulin 8 IGF2insulin growth factor 11p15.5 820 (X07868) G, A .46 9 9 PROS1 protein Salpha 3p11-cen 183 (M36564) A, G .50 10 10 LIPC triglyceride lipase15q21-q23 113 (M29189) T, G .49 11 11 CD18 integrin B-2 subunit 21q22.3109 (X64081) C, T .50 12 12 LDLR low density 19p13.2 70 (L00344) G, A.50 13 lipoprotein receptor The site numbers are specific single pointvariations located within the respective genes. All variations indicatedare defined on the sense strand. Genbank accession numbers are indicatedin parentheses. Chr., chromosome; Het., heterozygosity. References inTable 10 2 M. Armstrong, et al., “A Polymorphic Cfo I Site In Exon 6 ofthe Human Cytochrome P450 CYPD6 Gene Detected by the Polymerase ChainReaction,” Human Genetics 91: 616-17 (1993), which is herebyincorporated by reference. 3 S. C. Bock, et al., “Antithrombin III Utah:Proline-407 to Leucine Mutation in a Highly Conserved Region Near theInhibitor Reactive Site,” Biochemistry 28: 3628 (1991), which is herebyincorporated by reference. 4 G. Dewald, et al., “Polymorphism of HumanComplement Component C6: An Amino Acid Substitution (glu/ala) Within theSecond Thrombospondin Repeat Differentiates Between the Two CommonAllotypes C6 A and C6 B,” Biochem, Biophys. Res. Commun. 194: 458-64(1993), which is hereby incorporated by reference. 5 P. A. Velden, etal., “Amino Acid Dimorphism in IL1A is Detectable by PCR Amplification,”Hum. Mol. Genet. 2: 1753 (1993), which is hereby incorporated byreference. 6 R. M. Cawthon, et al., “Identification and Characterizationof Transcripts From Theneurofibromatosis 1 Region: The Sequence andGenomic Structure of EV12 and Mapping of Other Transcripts,” Genomics 7:555-65 (1990), which is hereby incorporated by reference. 7 C. C.Brooks, et al., “Association of the Widespread A149P Hereditary FructoseIntolerance Mutation With Newly Identified Sequence Polymorphisms in theAldolase B Gene,” Am. J. Human Genetics 52: 835-40 (1993), which ishereby incorporated by reference. 8 W. Poller, et al., “SequencePolymorphism in the Human Alpha-2-Macroglobulin (A2M) Gene,” NucleicAcids Res. 19: 198 (1991), which is hereby incorporated by reference. 9T. Gloudemans, “An Ava II Restriction Fragment Length Polymorphism inthe Insulin-Like Growth Factor II Gene and the Occurrence of SmoothMuscle Tumors,” Cancer Res. 53: 5754-58 (1993), which is herebyincorporated by reference. 10 C. M. Diepstraten, et al., “A CCA/CCGNeutral Dimorphism in the Codon for Pro 626 of the Human Protein S GenePSa (PROS1),” Nucleic Acids Res. 19: 5091 (1991), which is herebyincorporated by reference. 11 M. Reina, et al., “SSCP Polymorphism inthe Human Hepatic Triglyceride Lipase (LIPC) Gene,” Hum. Mol. Genet. 1:453 (1992), which is hereby incorporated by reference. 12 S. Mastuura,et al., “Investigation of the Polymorphic AvaII Site by a PCR-basedAssay at the Human CD18 Gene Locus,” Human Genetics 93: 721 (1994),which is hereby incorporated by reference. 13 L. Warnich, et al.,“Detection of a Frequent Polymorphism in Exon 10 of the Low-DensityLipoprotein Receptor Gene,” Human Genetics 89: 362 (1992), which ishereby incorporated by reference.

Each region was well characterized and harbored a single-base variationwith only two known alleles. PCR amplifications were performed usinggenomic DNA isolated from whole blood using the Purgene DNA IsolationKit (Gentra Systems, Inc., Minneapolis, Minn.) according to themanufacturer's instructions.

A volume of 25 μl of PCR buffer (10 mM Tris/HCl pH 8.3, 10 mM KCl, 4 mMMgCl₂, 0.4 mM each dNTP), 10-100 ng of genomic target DNA, PCR hybridprimer pairs 1-12 (2 pmol of each primer), and 1.3 units of AmpliTaq DNApolymerase Stoffel fragment (Applied Biosystems) was placed in athin-walled MicroAmp reaction tube (Applied Biosystems). Each hybridprimer consisted of a gene-specific 3′ region (16-29 bases) and a 5′region (22 bases) corresponding to one of two sets of universal (i.e.the portions which are primer-specific) primers (See FIG. 4, whereF1=Tet, F2=Fam, and F3=Het). These primers are shown in Table 11.

TABLE 11 Primary PCR Primer Sequences Number or Primer Name (5′-->3′)  1F GGAGCACGCTATCCCGTTAGAC AGCCAAGGGGAACCCTGAGAG (SEQ. ID. NO. 49)R CGCTGCCAACTACCGCACTATG ATCGTGGTCGAGGTGGTCACCATC (SEQ. ID. NO. 50)  2F CCTCGTTGCGAGGCGTATTCTG TATTTCCTCTTCTGTAAAAGGGAAGTTTGT (SEQ. ID.NO. 51) R GCGACCTGACTTGCCGAAGAAC ATGTCCCATCTCCTCTACCTGATAC (SEQ. ID. NO.52)  3 F GGAGCACGCTATCCCGTTAGAC TAAAGATCTGTCTTGCGTCCCAGTCA (SEQ. ID. NO.53) R CGCTGCCAACTACCGCACTATG TATCAATTTTGCAGAGCTTAGATGGAATG (SEQ. ID.NO. 54)  4 F CCTCGTTGCGAGGCGTATTCTGTAGCACTTGTGATCATGGTTTTAGAAATC (SEQ. ID. NO. 1) R GCGACCTGACTTGCCGAAGAACTATCGTATTTGATGATCCTCATAAAGTTG (SEQ. ID. NO. 56)  5F GGAGCACGCTATCCCGTTAGAC ATCAGCCACTTGGAAGGAGCAAAC (SEQ. ID. NO. 57)R CGCTGCCAACTACCGCACTATG ATGGACCATGGCTGAGTCTCCTTTAG (SEQ. ID. NO. 58)  6F CCTCGTTGCGAGGCGTATTCTG AACCAACACGGAGAAGCATTGTTTTC (SEQ. ID. NO. 59)R GCGACCTGACTTGCCGAAGAAC TATTAGCCTCAATCCTCATACTGACCTCTAC (SEQ.ID. NO. 60)  7 FGGAGCACGCTATCCCGTTAGACATCTCCTAACATCTATGTACTGGATTATCTAAATG (SEQ. ID. NO. 61)R CGCTGCCAACTACCGCACTATG ATCTTACTCAAGTAATCACTCACCAGTGTTG (SEQ.ID. NO. 62)  8 F CCTCGTTGCGAGGCGTATTCTGAATGAGTCAAATTGGCCTGGACTTG (SEQ. ID. NO. 63) R GCGACCTGACTTGCCGAAGAACTTAATTCCCGTGAGAAGGGAGATG (SEQ. ID. NO. 64)  9 F CCTCGTTGCGAGGCGTATTCTGAAGGATCTGGATGAAGCCATTTCTAAAC (SEQ. ID. NO. 65) R GCGACCTGACTTGCCGAAGAACTTGGAAAAGGTATTATAAGCAGAGAAAAGATG (SEQ. ID. NO. 66) 10F GGAGCACGCTATCCCGTTAGAC AGGACCGCAAAAGGCTTTCATC (SEQ. ID. NO. 67)R CGCTGCCAACTACCGCACTATG TAGCACCCAGGCTGTACCCAATTAG (SEQ. ID. NO. 68) 11F CCTCGTTGCGAGGCGTATTCTG ATCGGGCGCTGGGCTTCAC (SEQ. ID. NO. 69)R GCGACCTGACTTGCCGAAGAAC ATCAGATGCCGCACTCCAAGAAG (SEQ. ID. NO. 70) 12F GGAGCACGCTATCCCGTTAGAC ATAAGAGCCCACGGCGTCTCTTC (SEQ. ID. NO. 1)R CGCTGCCAACTACCGCACTATGTAAGAGACAGTGCCCAGGACAGAGTC (SEQ. ID. NO. 72)ZipALg1 F GGAGCACGCTATCCCGTTAGAC (SEQ. ID. NO. 73) ZipBLg2R CGCTGCCAACTACCGCACAT G (SEQ. ID. NO. 74) ZipCLg3F CCTCGT GCGAGGCGTATTCT G (SEQ. ID. NO. 75) ZipDLg4R GCGACCTGACTTGCCGAAGAAC (SEQ. ID. NO. 76) Solid underline denotes theZipALg1 and ZipBLg2 sequences. Dotted underline denotes the ZipCLg1 andZipDLg2 sequences. Linker sequences are indicated in bold. F = forward,R = Reverse.Forward and reverse hybrid primers for loci 1, 3, 5, 7, 10, and 12contained 5′ end regions identical to universal primers ALg1 and BLg2respectively. Forward and reverse primers to loci 2, 4, 6, 8, 9, and 11contained 5′ end regions identical to universal primers CLg3 and DLg4,respectively. The rationale for using a low concentration of the hybridprimers in the PCR phase was to deplete the hybrid primers during thereaction. This would theoretically allow products with low amplificationefficiencies to “catch up with” those that had high amplificationefficiencies. Amplification was attained by thermal cycling for 1 cycleof 96° C. for 15 sec to denature, then 15 cycles of 94° C. for 15 sec todenature and 65° C. for 60 sec to anneal and extend.

An equal volume of PCR buffer containing a high concentration of the twopairs of universal primers (25 pmol of each primer; Table 2) and 1.3units of Amplitaq DNA polymerase Stoffel fragment was added to theMicroamp reaction tube, and thermal cycling was performed for another 25cycles with the annealing temperature lowered to 55° C. The upstreamuniversal primers ALg1 and CLg3 were fluorescently labeled with 6-FAMand TET, respectively. All thermal cycling was achieved with a GeneAmpPCR System 9600 thermal cycler (Applied Biosystems).

The primary PCR process was carried out under the following conditions:

5.9 μl H₂O 5 μl primer pairs 1-12 (2 pmol each primer) 2.5 μl 10XStoffel fragment 4 μl 25 mM MgCl₂ 5 μl 2 mM dNTP stock (each) 2.5 μlgenomic DNA (10 ng) 0.13 μl Stoffel Frag. (1.3 units) 25 μl Total

The primary PCR cycling conditions were as follows:

-   -   96° C. 15″    -   94° C. 15″, 65° C. 1′×15    -   65° C. Hold        The secondary PCR process was carried out under the following        conditions:

13.37 μl H₂O 5 μl zip primer pairs (25 pmol each primer) 2.5 μl 10XStoffel fragment 4 μl 25 mM MgCl₂ 0.13 μl Stoffel Frag. (1.3 units) 25μl TotalThe secondary PCR cycling conditions were as follows:

-   -   94° C. 15″, 55° C. 1′×25    -   4° C. Hold

The PCR products were separated on an Applied Biosystems 373 DNAsequencer. A 3 ml aliquot of PCR sample was mixed with 3 ml of formamidecontaining fluorescently labeled Genescan-2500 [TAMRA] size standard(Applied Biosystems). The formamide/standard solution was prepared byadding 50 ml Genescan-2500 size standard [TAMRA] to 450 ml of formamide.The sample was heated at 95° C. for 2 min, quick cooled in ice, andelectrophoresed through a denaturing 8% polyacrylamide gel in an AppliedBiosystems 373 DNA sequencer running Genescan version 1.2 software. Thesizes of the fluorescently labeled products were automatically computedby the Genescan analysis software using the local Southern method. Theelectropherograms clearly showed 12 distinct products (FIG. 28). Theuniform amount of each product was attributed to the similar size ofeach amplicon and the use of the primary primers with portionscomplementary to the secondary primers, which annealed with identicalaffinities to the 12 amplicons without the need to carefully adjustreaction conditions. The computed sizes of the products, which rangedfrom 135 to 175 bp, matched exactly to their actual sizes (see Table12).

TABLE 12 List of PCR and LDR Products Site PCR Products LDR ProductNumber Label Size (bp) Label Size (bp) Variation Allele 1 6-FAM 1436-FAM 49 C A TET T B 2 TET 145 6-FAM 35 C A TET T B 3 6-FAM 147 6-FAM 43A A TET C B 4 TET 149 6-FAM 51 T A TET G B 5 6-FAM 155 HEX 56 A A 58 G B6 TET 157 6-FAM 53 A A TET G B 7 6-FAM 159 6-FAM 47 G A TET A B 8 TET161 6-FAM 45 G A TET A B 9 TET 165 6-FAM 55 A A TET G B 10 6-FAM 167 HEX48 T A 50 G B 11 TET 173 HEX 44 C A 46 T B 12 6-FAM 175 HEX 40 G A 42 AB The dual labeling approach (Table 12) made it much easier todistinguish the products on the electropherograms (FIG. 28, comparepanel A with panels B and C).

Example 10 Multiplex Ligase Detection Reaction

To avoid any possibility of labeled PCR product interfering with thedetection of ligation product sequences, PCR product amplified usingunlabeled PCR universal primers served as the target for the LDR. Thepolymerase in the PCR was inactivated by either freeze-thawing or addingEDTA/proteinase K to a final concentration of 5 mM and 100 mg/ml,respectively, and heating to 37° C. for 30 min and 95° C. for 10 min.

Proteinase K digestion was carried out under the following conditions:

20 μl PCR product +2.5 μl 500 mg/ml proteinase K 25 μl Total 60° C. for60 min. 95° C. for 10 min to heat kill proteinase K.

Four microliters of PCR product was diluted in 20 μl of LDR mixcontaining 50 mM Tris/HCl pH 8.5, 50 mM KCl, 10 mM MgCl₂, 1 mM NAD+, 10mM DTT, LDR oligonucleotide sets 1-12 (200 fmol of eacholigonucleotide), and 10 units of Thermus aquaticus DNA ligase (Barany,F. and Gelfand, D., “Cloning, Overexpression, and Nucleotide Sequence ofa Thermostable DNA Ligase-Encoding Gene,” Gene, 109:1-11 (1991), whichis hereby incorporated by reference). Each LDR oligonucleotide probe setconsisted of two allele-specific oligonucleotides and a commonoligonucleotide. Each pair of discriminating allele-specificoligonucleotide probes in the LDR oligonucleotide probe sets 1, 2, 3, 4,6, 7, 8, and 9 were the same size, with one oligonucleotide labeled with6-FAM and the other labeled with TET. For LDR oligonucleotide probe sets5, 10, 11, and 12, each pair of allele-specific oligonucleotidesdiffered by 2 bases (the larger oligonucleotide had a 5′ tail that wasnot complementary to the target sequence), and both oligonucleotideswere labeled with HEX. Thermal cycling was performed for 1 cycle of 95°C. for 2 min to denature, then 20 cycles of 95° C. for 30 sec todenature, and 65° C. for 4 min to ligate.

The LDR process was carried out as follows:

4 μl PCR product 2 μl LDR oligo sets 1-12 (200 fmol each oligo) 2 μl 10XT. ligase buffer 2 μl 10 mM NAD+ 1 μl 200 mM DTT 9 μl H₂O 20 μl Total +1μl Taq Ligase (10 units) Stock of Ligase: 0.5 μl of 625 u/ml + 3.2 μl10X buffer + 27.5 μl H₂0LDR cycling conditions were as follows:

-   -   94° C. 2′    -   94° C. 30″, 65° C. 4′×20    -   4° C. Hold

A 3 μl aliquot of LDR sample was mixed with 3 μl of formamide containingfluorescently labeled Genescan-2500 [TAMRA] size standard (AppliedBiosystems). The sample was heated at 95° C. for 2 min, quick cooled inice, and electrophoresed through a denaturing 10% polyacrylamide gel inan Applied Biosystems 373 DNA sequencer running Genescan version 1.2software. The sizes of the fluorescently labeled products wereautomatically computed by the Genescan analysis software using the localSouthern method. LDR profiles of two individuals are shown in FIG. 29.When each fluorescent dye was analyzed independently (FIG. 29, panelsB-D and F-H), it was very easy to determine the alleles present for eachlocus. A simple “A” or “B” code was assigned to each LDR product (Table12) and used to score the genotypes. See Table 13 as follows:

TABLE 13 Genotypes Determined by PCR-LDR for 5 Individuals IndividualSite Number 1 2 3 4 5 6 7 8 9 10 11 12 1 AB AB AB AB AB AB AB AA AA ABAB AB 2 AA BB AB BB AB AB BB AA AB AA AA AA 3 AB AB AB BB AA BB BB AA AAAB AB AA 4 AA AB AA AB AB AA AB AA AB BB AB AA 5 AA AA AA AB AB AB AB ABAA AB AB BBThe first individual (FIG. 29A-D; Table 13, individual 1) washeterozygous at polymorphic sites 1-7, and 10-12. Heterozygosity atsites 1-4 and 6-7 was indicated by the detection of both 6-FAM and TETlabeled products (FIG. 29, panels B and C) at the respective positionson the electropherograms. Heterozygosity at sites 5 and 11-12 wasindicated by the presence of two HEX labeled products, differing in sizeby 2 bases, for each of these loci (FIG. 29, panel D). In contrast, theone product detected at sites 8 and 9 (FIG. 29, panels B and C)established that each of these loci was homozygous. The secondindividual (FIG. 29E-F and G-H; Table 13, individual 2) was heterozygousonly at sites 3, 5, 6, and 9 and homozygous at sites 1, 2, 4, 7, 8,10-12. There was a total of 8 differences in the genotypes at thesepositions between the two persons. Three additional individuals weretyped, and all 5 persons had distinct genotypes based on the 12 loci(Table 13).

Although the invention has been described in detail for the purpose ofillustration, it is understood that such details are solely for thatpurpose. The variations can be made therein by those skilled in the artwithout departing from the spirit of the scope of the invention which isdefined by the following claims.

1. A method comprising: providing a sample potentially containing one ormore target nucleotide sequences; providing one or more primaryoligonucleotide primer sets, each set characterized by (a) a firstoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primer-specific portion, and (b) a secondoligonucleotide primer, having a target-specific portion and a 5′upstream secondary primer-specific portion; subjecting a primarypolymerase chain reaction mixture, comprising the sample, the one ormore primary oligonucleotide primer sets, and a polymerase, to two ormore polymerase chain reaction cycles to form primary extensionproducts; providing one or a plurality of secondary oligonucleotideprimer sets, each set characterized by (a) a first secondary primercontaining the same sequence as the 5′ upstream portion of a firstprimary oligonucleotide primer, and (b) a second secondary primercontaining the same sequence as the 5′ upstream portion of a secondprimary oligonucleotide primer from the same primary oligonucleotideprimer set as the first primary oligonucleotide primer contained by thefirst secondary primer; and subjecting a secondary polymerase chainreaction mixture, comprising the primary extension products, the one orplurality of secondary oligonucleotide primer sets, and the polymeraseto two or more polymerase chain reaction cycles to form secondaryextension products complementary to the primary extension products. 2.The method of claim 1 further comprising: sequencing the secondaryextension products.
 3. The method of claim 1 further comprising:coupling a label to the secondary extension products and detecting thelabel coupled to the secondary extension products.
 4. The method ofclaim 3, wherein said detecting comprises: distinguishing two or more ofa plurality of target nucleotide sequences in the sample that differ byone or more single-base changes, insertions, deletion, ortranslocations.
 5. The method of claim 3, wherein said detectingcomprises: sequencing the secondary extension products.
 6. The method ofclaim 1, wherein one of the primary oligonucleotide primers of a primaryoligonucleotide primer set comprises a further portion.
 7. The method ofclaim 6, wherein the further portion of one primary primer in a primaryoligonucleotide primer set differs from the further portion of a primaryprimer is a different primary oligonucleotide primer set.
 8. The methodof claim 1, wherein the secondary extension products are immobilized ona solid support.
 9. A method comprising: providing a sample potentiallycontaining one or more target nucleotide sequences; forming primaryextension products complementary to each of the one or more targetnucleotide sequences in a polymerase reaction mixture, wherein eachprimary extension product comprises an upstream primer portion, atarget-specific portion, and a downstream primer portion, wherein theupstream primer portion and the downstream primer portion are not thesame as or complementary to the target nucleotide sequence; and formingsecondary extension products that are complementary to each of theprimary extension products in a secondary polymerase reaction mixture.10. The method of claim 9 further comprising: sequencing the secondaryextension products.
 11. The method of claim 9, wherein each secondaryextension product comprises an upstream primer portion, a targetspecific portion, and a downstream target specific portion.
 12. Themethod of claim 9 further comprising: coupling a label to the secondaryextension products and detecting the label coupled to the secondaryextension products formed in the secondary polymerase chain reactionmixture.
 13. The method of claim 12, wherein said detecting comprises:distinguishing two or more target nucleotide sequences in the samplethat differ by one or more single-base changes, insertions, deletion, ortranslocations.
 14. The method of claim 12, wherein said detectingcomprises: sequencing the secondary extension products.
 15. The methodof claim 9, wherein the primary extension products comprise a furtherportion.
 16. The method of claim 15, wherein the further portion of oneprimary extension product differs from the further portion of adifferent primary extension product.
 17. The method of claim 9, whereinthe secondary extension products are immobilized on a solid support.